PubMed Health⌕ Search

Biomedical subjects

T Ashley

Publications and source records attributed to T Ashley.

At least 37 records · Page 2Linked to original sources
PubMed1997

Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes.

BACKGROUND: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest. RESULTS: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22-23, a region marked by frequent deletions and loss of heterozygosity in human tumors. CONCLUSIONS: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.

Amino Acid Sequence↗
PubMed1997

The telomere-associated DNA from human chromosome 20p contains a pseudotelomere structure and shares sequences with the subtelomeric regions of 4q and 18p.

The human chromosome 20p telomere has been cloned on a yeast artificial chromosome (YAC). The telomere-associated DNA contains an interstitial tract of (TTAGGG)n telomeric repeats 60 kb in from the chromosome end. Frequent truncation of the YAC clone was observed due to resolution of the internal telomeric array into a telomere. The 20p internal telomeric repeat tract is flanked on its centromeric side by telomere-associated repeated sequences that have previously been found adjacent to terminal telomeric repeat arrays. The pseudotelomere structure of the 20p subtelomeric region is similar to the structure of some yeast subtelomeric regions where these sequences act as substrates for recombinational repair of chromosome ends that have lost their terminal telomeric repeat arrays. Sequences flanking the telomeric end of the internal (TTAGGG)n repeat array on 20p are found adjacent to three other subtelomeric (TTAGGG)n tracts on 4q, 18p, and an unknown chromosome end, respectively. These shared sequences provide evidence of exchange between nonhomologous chromosomes in humans.

Chromosomes, Artificial, Yeast↗
PubMed1997

Evidence for a role for DNA polymerase beta in mammalian meiosis.

DNA polymerase beta (pol beta) is an enzyme possessing both polymerase and deoxyribose phophatase activities. Although pol beta is not believed to participate in the replication of genomic DNA, several studies have indicated a role for pol beta in DNA repair. The high level of expression of pol beta in mouse and rat testes raises the possibility that pol beta participates in mammalian meiosis. Using antibody localization, we detect foci that stain with pol beta antisera at discrete sites along homologous chromosomes as they synapse and progress through prophase of meiosis I. These data suggest that pol beta participates in meiotic events associated with synapsis and recombination.

Animals↗
PubMed1997

Association of BRCA1 with Rad51 in mitotic and meiotic cells.

BRCA1 immunostaining reveals discrete, nuclear foci during S phase of the cell cycle. Human Rad51, a homolog of bacterial RecA, behaves similarly. The two proteins were found to colocalize in vivo and to coimmunoprecipitate. BRCA1 residues 758-1064 alone formed Rad51-containing complexes in vitro. Rad51 is also specifically associated with developing synaptonemal complexes in meiotic cells, and BRCA1 and Rad51 were both detected on asynapsed (axial) elements of human synaptonemal complexes. These findings suggest a functional interaction between BRCA1 and Rad51 in the meiotic and mitotic cell cycles, which, in turn, suggests a role for BRCA1 in the control of recombination and of genome integrity.

BRCA1 Protein↗
PubMed1997

ATM and RPA in meiotic chromosome synapsis and recombination.

ATM is a member of the phosphatidylinositol 3-kinase (PIK)-like kinases, some of which are active in regulating DNA damage-induced mitotic cell-cycle checkpoints. ATM also plays a role in meiosis. Spermatogenesis in Atm-/- male mice is disrupted, with chromosome fragmentation leading to meiotic arrest; in human patients with ataxia-telangiectasia (A-T), gonadal atrophy is common. Immuno-localization studies indicate that ATM is associated with sites along the synaptonemal complex (SC), the specialized structure along which meiotic recombination occurs. Recombination, preceded by pairing of homologous chromosomes, is thought to require heteroduplex formation between homologous DNA, followed by strand exchange. These early meiotic steps (entailing the formation and processing of meiotic recombination intermediates with DNA-strand interruptions) require ssDNA-binding proteins such as replication protein A (RPA; refs 5-7). In somatic cells, DNA damage induces ATM-dependent phosphorylation of RPA. We demonstrate here that ATM and RPA co-localize along synapsed meiotic chromosomes and at sites where interactions between ectopic homologous chromosome regions appear to initiate. In Atm-/- meiotic prophase spermatocytes, immuno-localization shows that RPA is present along synapsing chromosomes and at sites of fragmentation of the SC. These results suggest that RPA and ATM co-localize at sites where interhomologous-DNA interactions occur during meiotic prophase and where breaks associated with meiotic recombination take place after synapsis, implying a possible functional interaction between these two proteins.

Animals↗
PubMed1996

Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects, and thymic lymphoma.

ATM, the gene mutated in the inherited human disease ataxia-telangiectasia, is a member of a family of kinases involved in DNA metabolism and cell-cycle checkpoint control. To help clarify the physiological roles of the ATM protein, we disrupted the ATM gene in mice through homologous recombination. Initial evaluation of the ATM knockout animals indicates that inactivation of the mouse ATM gene recreates much of the phenotype of ataxia-telangiectasia. The homozygous mutant (ATM-/-) mice are viable, growth-retarded, and infertile. The infertility of ATM-/- mice results from meiotic failure. Meiosis is arrested at the zygotene/pachytene stage of prophase I as a result of abnormal chromosomal synapsis and subsequent chromosome fragmentation. Immune defects also are evident in ATM-/- mice, including reduced numbers of B220+CD43- pre-B cells, thymocytes, and peripheral T cells, as well as functional impairment of T-cell-dependent immune responses. The cerebella of ATM-/- mice appear normal by histologic examination at 3 to 4 months and the mice have no gross behavioral abnormalities. The majority of mutant mice rapidly develop thymic lymphomas and die before 4 months of age. These findings indicate that the ATM gene product plays an essential role in a diverse group of cellular processes, including meiosis, the normal growth of somatic tissues, immune development, and tumor suppression.

Animals↗
PubMed1996

The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes.

A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia- and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are approximately 300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination.

Animals↗
PubMed1996

Presynaptic association of Rad51 protein with selected sites in meiotic chromatin.

Eukaryotic homologs of Escherichia coli Rec-A protein have been shown to form nucleoprotein filaments with single-stranded DNA that recognize homologous sequences in duplex DNA. Several recent reports in four widely diverse species have demonstrated the association of RecA homologs with meiotic prophase chromatin. The current immunocytological study on mouse spermatocytes and oocytes shows that a eukaryotic homolog, Rad5l, associates with a subset of chromatin sites as early as premeiotic S phase, hours before either the appearance of precursors of synaptonemal complexes or the initiation of synapsis. When homologous chromosomes do begin to pair, the Rad5l-associated sequences are sites of initial contact between homologues and of localized DNA synthesis. Distribution of Rad5l foci on the chromatin of fully synapsed bivalents at early pachynema corresponds to an R-band pattern of mitotic chromosomes. R-bands are known to be preferred sites of both synaptic initiation and recombination. The time course of appearance of Rad51 association with chromatin, its distribution, and its interaction with other Rad5l-associated sequences suggests that it plays an important role preselection of sequences and synaptic initiation.

Animals↗
PubMed1996

Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

Amino Acid Sequence↗
PubMed1996

Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over.

Mice that are deficient in either the Pms2 or Msh2 DNA mismatch repair genes have microsatellite instability and a predisposition to tumours. Interestingly, Pms2-deficient males display sterility associated with abnormal chromosome pairing in meiosis. Here mice deficient in another mismatch repair gene, Mlh1, possess not only microsatellite instability but are also infertile (both males and females). Mlh1-deficient spermatocytes exhibit high levels of prematurely separated chromosomes and arrest in first division meiosis. We also show that Mlh1 appears to localize to sites of crossing over on meiotic chromosomes. Together these findings suggest that Mlh1 is involved in DNA mismatch repair and meiotic crossing over.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis.

Using gene targeting in embryonic stem cells, we have derived mice with a null mutation in a DNA mismatch repair gene homolog, PMS2. We observed microsatellite instability in the male germline, in tail, and in tumor DNA of PMS2-deficient animals. We therefore conclude that PMS2 is involved in DNA mismatch repair in a variety of tissues. PMS2-deficient animals appear prone to sarcomas and lymphomas. PMS2-deficient males are infertile, producing only abnormal spermatozoa. Analysis of axial element and synaptonemal complex formation during prophase of meiosis I indicates abnormalities in chromosome synapsis. These observations suggest links among mismatch repair, genetic recombination, and chromosome synapsis in meiosis.

Adenosine Triphosphatases↗
PubMed1995

Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates.

Antibodies against human Rad51 protein were used to examine the distribution of Rad51 on meiotic chromatin in mouse spermatocytes and oocytes as well as chicken oocytes during sequential stages of meiosis. We observed the following dynamic changes in distribution of Rad51 during meiosis: (1) in early leptotene nuclei there are multiple, apparently randomly distributed, foci that by late leptonema become organized into tracks of foci. (2) These foci persist into zygonema, but most foci are now localized on Rad51-positive axes that correspond to lateral elements of the synaptonemal complex. As homologs synapse foci from homologous axes fuse. The distribution and involvement of Rad51 foci as contact points between homologs suggest that they may be components to early recombination nodules. (3) As pachynema progresses the number of foci drops dramatically; the temporal occurrence (mice) and physical and numerical distribution of foci on axes (chickens) suggest that they may be a component of late recombination nodules. (4) In early pachynema there are numerous Rad51 foci on the single axis of the X (mouse spermatocytes) or the Z (chicken oocytes) chromosomes that neither pair, nor recombine. (5) In late pachynema in mouse spermatocytes, but not oocytes, the Rad51 signal is preferentially enhanced at both ends of all the bivalents. As bivalents in spermatocytes, but not oocytes, begin to desynapse at diplonema they are often held together at these Rad51-positive termini. These observations parallel observations that recombination rates are exceptionally high near chromosome ends in male but not female eutherian mammals. (6) From diakinesis through metaphase I, Rad51 protein is detected as low-intensity fluorescent doublets that localize with CREST-specific antigens (kinetochores), suggesting that Rad51 participates, at least as a structural component of the materials involved, in sister kinetochore cohesiveness. Finally, the changes in Rad51 distribution during meiosis do not appear to be species specific, but intrinsic to the meiotic process.

Animals↗
PubMed1995

Multicolor FISH with a telomere repeat and Sry sequences shows that Sxr (Sex reversal) in the mouse is a new type of chromosome rearrangement.

XYSxr (Sex reversal) mice carry a Y chromosome in which the chromatin (including Sry, the gene for testis determination) that normally resides on the short arm is duplicated and the second copy is relocated to the distal end of the long arm. Multicolor in situ hybridization to mitotic chromosomes of XYSxr males using probes for the telomere repeat sequence (TTAGGG)n and Sry shows that the rearranged chromatin is located distal to the telomeric signal. This suggests that the rearrangement arose from a recombination event involving the distal Y telomere sequences, i.e., within the telomere, a structure historically assumed to be incapable of participating in chromosome rearrangements.

Animals↗
PubMed1994

Detection of nondisjunction and recombination in meiotic and postmeiotic cells from XYSxr [XY,Tp(Y)1Ct] mice using multicolor fluorescence in situ hybridization.

Current meiotic dogma holds that synapsis is required for recombination and that recombination is required for proper disjunction. The mouse chromosome aberration XYSxr [sex reversal; redesignated XY,Tp(Y)1Ct] appears to challenge this assumption, for although chromosomes X and Y often fail to synapse and recombine, there is no dramatic increase in aneuploid progeny. An explanation of this conundrum might be that X-Y univalent spermatocytes do not survive. The phenotype of sex reversal is generated by the "obligatory" crossover between the X and Y chromosomes, which always occurs proximal to a duplicated copy of the testis-determining gene Sry and transfers one copy from one chromatid of the Y chromosome to one chromatid of the X. Animals that inherit an X chromosome with the Sry gene are chromosomally female but phenotypically male. We have used fluorescence in situ hybridization (FISH) to visualize probes for the X and Y chromosomes and for the Sry sequence and chromosome 8 to track the fate of both recombinant and nonrecombinant chromosomes through metaphases I and II into spermatids and sperm. In the 219 gametes examined by multicolor FISH, the frequency of aneuploid products (XY or "O") was low (3.7%) despite a high frequency (66%) of X-Y separation at metaphase I. In balanced gametes, X and Y recombinant chromosomes slightly exceeded nonrecombinants. Both of these observations support the earlier proposal that asynapsis and nondisjunction in primary spermatocytes lead to their developmental arrest and degeneration.

Aneuploidy↗
PubMed1994

Mammalian meiotic recombination: a reexamination.

Recombination nodules (RNs) are small electron-dense structures associated with the synaptonemal complex. Two types have been identified: early RNs present during zygonema-early pachynema, which are thought to be involved in gene conversion and synaptic initiation, and late RNs present during mid-to-late pachynema, which are thought to be involved in reciprocal recombination leading to chiasma formation. In organisms as diverse as Sodaria, Drosophila, and plants there is indeed a close correlation between the observed number of late RNs and crossovers, or their cytogenetic manifestation, chiasmata. However, as this reexamination of the human data shows, there is not a similar correlation in mammals. Instead, there is a severe deficiency in RNs in eutherian males and marsupial females near chromosome ends and other recombinational "hot spots" (defined genetically), or "localized chiasmata" (defined cytogenetically). Many of these sites of hyper-recombination correspond to sites of telomere or telomere-associated sequences. Together these observations suggest the possibility of a second, mechanistically different, recombination pathway that does not involve RNs, but may directly involve telomere or telomere-associated sequences. This pathway may be responsible for sex-specific hot-spots of recombination observed at highly localized sites throughout the genome.

Animals↗
PubMed1994

The curious normality of the synaptic association between the sex chromosomes of two arvicoline rodents: Microtus oeconomus and Clethrionomys glareolus.

In all eight species of arvicoline (microtine) rodents previously described, the X and Y chromosomes have remained asynaptic throughout pachynema. Since synapsis is presumed to be a prerequisite for crossing over, it has been concluded that the sex chromosomes in these species are also achiasmatic, but the mechanism(s) of their disjunction remains an enigma. Their asynaptic, achiasmatic condition has been attributed to loss of the pseudoautosomal region (Borodin et al. 1991; Carnero et al. 1991; Jiménez et al. 1991). This loss has been postulated to include all arvicoline rodents. We describe here the sex chromosome behavior during meiotic prophase of two additional species in this group: Microtus oeconomus and Clethrionomys glareolus. In both species there is extensive synapsis between the X and Y, providing the usual opportunity for XY recombination. These findings challenge the concept of the pseudoautosomal region as an evolutionarily conserved region of homology, at least within the arvicoline rodents. The unexpected finding of synapsis in two very different species, one with a derived and one with a primitive karyotype is discussed within its phylogenetic context.

Animals↗
PubMed1993

Molecular characterization of a pericentric inversion in mouse chromosome 8 implicates telomeres as promoters of meiotic recombination.

A "hot spot" of meiotic recombination has been found in males on murine chromosome 8 using nonisotopic hybridization of a series of probes to mitotic and meiotic chromosomes. The sequences responsible for this enhanced recombination are the telomeric repeats. Mice both normal and hetero- or homozygous for a pericentric inversion, In(8)1 Rl, were analyzed. The inversion subdivides chromosome 8 into three discreet regions: (1) a fraction of the micro "short arm" that contains 30-150 kb of telomeric sequences and only about one-fifth of the contiguous minor-satellite sequences (approximately 200 kb); (2) the inverted region; and (3) the noninverted distal two-thirds of the chromosome. In 70 spermatocytes from inversion heterozygotes, examined by electron microscopy, synapsis of the inverted region was complete but entirely nonhomologous. Nonhomologous synapsis persists from initiation of synaptonemal complex formation in zygonema/early pachynema until dissolution in late pachynema. This nonhomologous synapsis also suppresses crossing over within the inverted segment. The opportunity for proximal homologous recombination is thus restricted to the roughly 250 kb segment located between the short-arm break and the end of the bivalent. Nonetheless, an extreme proximal chiasma was observed in 11% of the heterozygous chromosome-8 bivalents, 34% of the normal 8 bivalents and 35% of the homozygous inversion 8 bivalents from spermatocyte preparations. Since in the normal chromosomes all minor satellite sequences are adjacent to the telomere, while in the inversion chromosomes most of these sequences are transposed to an interstitial position without a corresponding shift in chiasma position, the minor-satellite sequences can be ruled out as promoters of recombination.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗