Molecular events associated with trisomy of chromosome 7 in mouse skin chemical carcinogenesis

The primary objective of this study has been to investigate the effects at the molecular level of trisomy of mouse chromosome 7 in chemically induced skin tumors. It was previously proposed that the initiation event in the mouse skin carcinogenesis model is a heterozygous mutation of the Ha-ras-1 gene, mapped to chromosome 7. Previous studies in this laboratory identified trisomy 7 as one of the primary nonrandom cytogenetic abnormalities found in the majority of severely dysplastic papillomas and squamous cell carcinomas induced in SENCAR mice by an initiation-promotion protocol. Therefore, the first hypothesis tested was that trisomy 7 occurs by specific duplication of the chromosome carrying a mutated Ha-ras-1 allele. Results of a quantitative analysis of normal/mutated allelic ratios of the Ha-ras-1 gene confirmed this hypothesis, showing that most of the tumors exhibited overrepresentation of the mutated allele in the form of 1/2, 0/3, and 0/2 (normal/mutated) ratios. In addition, histopathological analysis of the tumors showed an apparent association between the degree of malignancy and the dosage of the mutated Ha-ras-1 allele. To determine the mechanism for loss of the normal Ha-ras-1 allele, found in 30% of the tumors, a comparison of constitutional and tumor genotypes was performed at different informative loci of chromosome 7. By combining Southern blot and polymerase chain reaction fragment length polymorphism analyses of DNAs extracted from squamous cell carcinomas, complete loss of heterozygosity was detected in 15 of 20 tumors at the Hbb locus, and in 5 of 5 tumors at the int-2 locus, both distal to Ha-ras-1. In addition, polymerase chain reaction analysis of DNA extracted from papillomas indicated that loss of heterozygosity occurs in late-stage lesions exhibiting a high degree of dysplasia and areas of microinvasion, suggesting that this event may be associated to the acquisition of the malignant phenotype. Allelic dosage analysis of tumors that had become homozygous at Hbb but retained heterozygosis at Ha-ras-1, indicated that loss of heterozygosity on mouse chromosome 7 occurs by a mitotic recombination mechanism. Overall, these findings suggest the presence of a putative tumor suppressor locus on the 7F1-ter region of mouse chromosome 7. Thus, loss of function by homozygosis at this putative suppressor locus may complement activation of the Ha-ras-1 gene during tumor progression, and might be associated with the malignant conversion stage of mouse skin carcinogenesis. ^

The functional and structural interactions between v-{\it mos\/} proteins and cell cycle control elements

The v-mos gene of Moloney murine sarcoma virus (Mo-MuSv) encodes a serine/threonine protein kinase capable of inducing cellular transformation. The c-mos protein is an important cell cycle regulator that functions during meiotic cell division cycles in germ cells. The overall function of c-mos in controlling meiosis is becoming better understood but the role of v-mos in malignant transformation of cells is largely unknown.^ In this study, v-mos protein was shown to be phosphorylated by M phase kinase in vitro and in vivo. The kinase activity and neoplastic transforming ability of v-mos is positively regulated by the phosphorylation. Together with the earlier finding of activation of M phase kinase by c-mos, these results raise the possibility of mutual regulation between M phase kinase and mos kinases.^ In addition to its functional interaction with the M phase kinase, the v-mos protein was shown to be present in the same protein complex with a cyclin-dependent kinase (cdk). In addition, an antibody that recognizes the cdk proteins was shown to co-precipitate the v-mos proteins in the interphase and mitotic cells transformed by p85$\sp{\rm gag-mos}$. Cdk proteins have been shown to be associated with nonmitotic cyclins which are potential oncogenes. The perturbation of cdk kinase or the activation of non-mitotic cyclins as oncogenes by v-mos could contribute directly to v-mos induced cellular transformation. v-mos proteins were also shown to interact with tubulin and vimentin, the essential components of microtubules and type IV intermediate filaments, respectively. The organizations of both microtubules and intermediate filaments are cell cycle-regulated. These results suggest that the v-mos kinase could be directly involved in inducing morphological changes typically seen in transformed cells.^ The interactions between the v-mos protein and these cell cycle control elements in regards to v-mos induced neoplastic transformation are discussed in detail in the text. ^

c-{\it mos\/} RNA expression in somatic cells and its physiological significance in cell cycle progression

The c-mos proto-oncogene, which is expressed at relatively high levels in male and female germ cells, plays a key role in oocyte meiotic maturation. The c-mos gene product in oocytes (p39$\sp{\rm c-mos}$) is necessary and sufficient to initiate meiosis. p39$\sp{\rm c-mos}$ is also an essential component of the cytostatic factor, which is responsible for arresting vertebrate oocytes at the second meiotic metaphase by stabilizing the maturation promoting factor (MPF). MPF is a universal regulator of both meiosis and mitosis. Much less is understood about c-mos expression and function in somatic cells. In addition to gonadal tissues, c-Mos has been detected in some somatic tissues and non-germ cell lines including NIH 3T3 cells as a protein termed p43$\sp{\rm c-mos}$. Since c-mos RNA transcripts were not previously detected in this cell line by Northern blot or S1 protection analyses, a search was made for c-mos RNA in NIH 3T3 cells. c-mos transcripts were detected using the highly sensitive RNA-PCR method and RNase protection assays. Furthermore, cell cycle analyses indicated that expression of c-mos RNA is tightly controlled in a cell cycle dependent manner with highest levels of transcripts (approximately 5 copies/cell) during the G2 phase.^ In order to determine the physiological significance of c-mos RNA expression in somatic cells, antisense mos was placed under the control of an inducible promoter and introduced into either NIH 3T3 cells or C2 cells. It was found that a basal level of expression of antisense mos resulted in interference with mitotic progression and growth arrest. Several nuclear abnormalities were observed, especially the appearance of binucleated and multinucleated cells as well as the extrusion of microvesicles containing cellular material. These results indicate that antisense mos expression results in a block in cytokinesis. In summary, these results establish that c-mos expression is not restricted to germ cells, but instead indicate that c-mos RNA expression occurs during the G2 stage of the cell cycle. Furthermore, these studies demonstrate that the c-mos proto-oncogene plays an important role in cell cycle progression. As in meiosis, c-mos may have a similar but not identical function in regulating cell cycle events in somatic cells, particularly in controlling mitotic progression via activation/stabilization of MPF. ^

Role of {\it ERCC1\/} in DNA repair and genetic recombination

The ERCC1 (Excision Repair Cross-Complementing-1) gene is the presumptive mammalian homolog of the Saccharomyces cerevisiae RAD10 gene. In mammalian NER, the Ercc1/XpF complex functions as an endonuclease that specifically recognizes 5$\sp\prime$ double-strand-3$\sp\prime$ single-strand structures. In yeast, the analogous function is performed by the Rad1/Rad10 complex. These observations and the conservation of amino acid homology between the Rad1 and XpF and the Rad10 and Ercc1 proteins has led to a general assumption of functional homology between these genes.^ In addition to NER, the Rad1/Rad10 endonuclease complex is also required in certain specialized mitotic recombination pathways in yeast. However, a similiar requirement for the endonuclease function of the Ercc1/XpF complex during genetic recombination in mammalian cells has not been directly demonstrated. The experiments performed in these studies were designed to determine if ERCC1 deficiency would produce recombination-deficient phenotypes in CHO cells similar to those observed in RAD10 deletion mutants, including: (1) decreased single-reciprocal exchange recombination, and (2) inability to process 5$\sp\prime$ sequence heterology in recombination intermediates.^ Specifically, these studies describe: (1) The isolation and characterization of the ERCC1 locus of Chinese hamster ovary cells; (2) The production of an ERCC1 null mutant cell line by targeted knock-out of the endogenous ERCC1 gene in a Chinese hamster ovary cell line, CHO-ATS49tg, which contains an endogenous locus, APRT, suitable as a chromosomal target for homologous recombination; (3) The characterization of mutant ERCC1 alleles from a panel of Chinese hamster ovary cell ERCC1 mutants derived by conventional mutagenesis; (4) An investigation of the effects of ERCC1 mutation on mitotic recombination through targeting of the APRT locus in an ERCC1 null background.^ The results of these studies strongly suggest that the role of ERCC1 in homologous recombination in mammalian cells is analogous to that of the yeast RAD10 gene. ^

Cloning and genetic characterization of Skb1: A novel regulator of Shk1 and mitosis in Schizosaccharomyces pombe

A partial skb1 gene was originally isolated in a yeast two-hybrid screen for Shk1-interacting polypeptides. Shk1 is one of two Schizosaccharomyces pombe p21Cdc42/Rac-activated kinases (PAKs) and is an essential component of the Ras1-dependent signal transduction pathways regulating cell morphology and mating responses in fission yeast. After cloning the skb1 gene we found the Skb1 gene product to be a novel, nonessential protein lacking homology to previously characterized proteins. However the identification of Skb1 homologs in C. elegans, S. cerevisiae, and H. sapiens reveals evolution has conserved the skb1 gene. Fission yeast cells carrying a deletion of skb1 exhibit a defect in cell size but not mating abilities. This defect is suppressed by high copy shk1. Fission yeast overexpressing skb1 were found to undergo cell division at a length 1.5X greater than normal. In the two-hybrid system, Skb1 interacts with a subdomain of the Shk1 regulatory region distinct from that with which Cdc42 interacts, and forms a ternary complex with Shk1 and Cdc42. By use of yeast genetics, we have established a role for Skb1 as a positive regulator of Shk1. Co-overexpression of shk1 with skb1 was found to suppress the morphology defect, but not the sterility, of ras1Δ fission yeast. Thus, the function of Skb1 is restricted to a morphology control pathway. We determined that Skb1 functions as a negative regulator of mitosis and does this through a Shk1-dependent mechanism. The mitotic regulatory function of Skb1 and Shk1 was also partially dependent upon Wee1, a direct negative regulator of the cyclin-dependent kinase Cdc2. The role for Skb1 and Shk1 as mitotic regulators is the first connection from a PAK protein to control of the cell cycle. Furthermore, Skb1 is the first non-Cdc42/Rac PAK modulator to be identified. ^

PREMATURE CONDENSATION OF SPERMATOGENIC CELL CHROMOSOMES

The technique of premature chromosome condensation (PCC) has been used primarily to study interphase chromosomes of somatic cells. In this study, mitotic cells were fused to cells from the mouse testes to examine the chromosomes of germ cells. The testes contain various types of cells, both germinal and nongerminal. In these initial studies, four types of PCC morphologies were observed. Chromosome morphology of the PCC and labeling experiments demonstrated the mouse cell origin of various PCC. Attempts were next made to determine the cell types producing the PCC. Spermatogonia, diplotene spermatocytes, secondary spermatocytes and round spermatids are proposed to be the origin of the PCC morphologies. Some PCC could be banded by G and C banding techniques and the mouse chromosomes identified.^ Studies were subsequently undertaken to evaluate this technique as a method of evaluating damage to germ cells. Testicular cells from irradiated mice were fused to mitotic cells and the PCC examined. Both round spermatids and secondary spermatocytes exhibited chromosome damage in the form of chromatid breaks. A linear correlation was found between the dose of irradiation and the number of breaks per cell. This technique may develop into a useful method for evaluating the clastogenic effect of agents on the germ cells. ^

CHROMOSOMAL ORIGIN OF DM-DNA

Double minutes (dm) are small chromatin particles of 0.3 microns diameter found only in the metaphase cells of human and murine tumors. Dm are unique cytogenetic structures since their numbers per cell show wide variation. At cell division, dm are retained despite the lack of centromeres. In squash preparations, dm show clustering often in association with chromosomes. Human carcinoma cell line SW613-S18 was found to have large numbers of dm and biological characteristics favorable for mitotic synchronization and chromosome isolation experiments.^ S18 cells were synchronized to mitosis with metabolic and mitotic blocking compounds. Mitotic cells were lysed to release chromosomes and dm from the mitotic spindle and the resulting suspensions were fractionated to enrich for dm. The DNA in enriched fractions was characterized. The reassociation kinetics of dm-DNA driven with placental human DNA was similar to the reassociation curve of labeled placental DNA under similar conditions. In situ hybridization of dm-DNA to tumor and normal metaphase cells showed grain localization over the entire karyotype. Dm-DNA was shown by pulse chase DNA replication experiments to replicate during early and mid S-phase of the cell cycle, but not in late S-phase. In addition, BrdUrd incorporation studies showed that dm-DNA replicates only once during the S-phase. Premature chromosome condensation studies suggest the basis of numerical heterogeneity of dm is nondisjunction, not anomalous or unscheduled DNA replication.^ These data and previous cytochemical banding studies of dm in SW613-S18 indicate that dm-DNA is chromosomal in origin. No evidence of gene amplification was found in the DNA reassociation data. It is likely that dm-DNA represents the pale-staining G-band regions of the human karyotype in this cell line. ^

CYTOGENETIC AND DNA CONTENT ANALYSIS IN MULTIPLE MYELOMA

In this dissertation, the cytogenetic characteristics of bone marrow cells from 41 multiple myeloma patients were investigated. These cytogenetic data were correlated with the total DNA content as measured by flow cytometry. Both the cytogenetic information and DNA content were then correlated with clinical data to determine if diagnosis and prognosis of multiple myeloma could be improved.^ One hundred percent of the patients demonstrated abnormal chromosome numbers per metaphase. The average chromosome number per metaphase ranged from 42 to 49.9, with a mean of 44.99. The percent hypodiploidy ranged from 0-100% and the percent hyperdiploidy from 0-53%. Detailed cytogenetic analyses were very difficult to perform because of the paucity of mitotic figures and the poor chromosome morphology. Thus, detailed chromosome banding analysis on these patients was impossible.^ Thirty seven percent of the patients had normal total DNA content, whereas 63% had abnormal amounts of DNA (one patient with less than normal amounts and 25 patients with greater than normal amounts of DNA).^ Several clinical parameters were used in the statistical analyses: tumor burden, patient status at biopsy, patient response status, past therapy, type of treatment and percent plasma cells. Only among these clinical parameters were any statistically significant correlations found: pretreatment tumor burden versus patient response, patient biopsy status versus patient response and past therapy versus patient response.^ No correlations were found between percent hypodiploid, diploid, hyperdiploid or DNA content, and the patient response status, nor were any found between those patients with: (a) normal plasma cells, low pretreatment tumor mass burden and more than 50% of the analyzed metaphases with 46 chromosomes; (b) normal amounts of DNA, low pretreatment tumor mass burden and more than 50% of the metaphases with 46 chromosomes; (c) normal amounts of DNA and normal quantities of plasma cells; (d) abnormal amounts of DNA, abnormal amounts of plasma cells, high pretreatment tumor mass burden and less than 50% of the metaphases with 46 chromosomes.^ Technical drawbacks of both cytogenetic and DNA content analysis in these multiple myeloma patients are discussed along with the lack of correlations between DNA content and chromosome number. Refined chromosome banding analysis awaits technical improvements before we can understand which chromosome material (if any) makes up the "extra" amounts of DNA in these patients. None of the correlations tested can be used as diagnostic or prognostic aids for multiple myeloma. ^

STUDIES ON THE BIOLOGICAL ACTIVITY OF MACROMOLECULES MICROINJECTED INTO MAMMALIAN SOMATIC CELLS

Red Blood cell mediated and glass needle mediated microinjection technology was used to introduce macromolecules into mammalian somatic cells. The biological activities of DNA synthesis inducing factor(s) (Chapter 1), mitotic factor(s) (Chapter 2), and DNA coding for ovalbumin and thymidine kinase (Chapter 3) were studied following injection into mammalian somatic cells.^ Chapter 1. A cell undergoing DNA replication (S phase) contains a factor(s) that induces DNA synthesis prematurely in a G(,1) nucleus when an S phase cell is fused to a G(,1) cell. An assay for the active factor(s) was developed in which a mixture of s phase extract loaded red blood cells (RBC) and synchronous G(,1) HeLa cells was centrifuged onto Concanavalin A (Con A) treated coverslips and fused by PEG. This technique is called "Centrifusion". The synchronous G(,1) HeLa cells injected with S phase extract initiated DNA synthesis earlier than the control G(,1) cells mock injected with RBC loaded with buffer.^ Chapter 2. It has been demonstrated that fusion between a mitotic and an interphase cell usually leads to breakdown of the interphase nucleus, followed by condensation of the interphase chromatin into discrete chromosomes, a process termed premature chromosome condensation. I wanted to develop an assay for the mitotic factor(s) that induces premature chromosome condensation. Experiments were performed utilizing glass needle mediated microinjection of HeLa cell mitotic extract into interphase somatic mammalian cells in an attempt to induce premature chromosome condensation. However, I was not able to induce premature chromosome condensation in the interphase cells, probably because of an inability to introduce sufficient mitotic factor(s) into the cells.^ Chapter 3. A recombinant plasmid containing the chicken ovalbumin gene and three copies of the Herpes thymidine Kinase gene (pOV12-TK) was introduced into mouse LMTK('-) cell nuclei using glass needle mediated gene transfer resulting in LMTK('+) clones that were selected for in HAT medium. Restriction enzyme analysis of the high molecular weight DNA from 6 HAT medium survivor cell clones revealed the presence of one or at best only a few copies of the 12kb ovalbumin gene per mouse genome. Further analysis showed the ovalbumin DNA was not rearranged and was associated with high molecular weight mouse cell DNA. Each of the analyzed cell clones produced ovalbumin demonstrating that the biological activity of the microinjected ovalbumin was retained. ^

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

STUDIES OF THE MOLECULAR MECHANISMS OF CHROMOSOME ABERRATION FORMATION (DNA REPAIR, CROSSLINKS, MITOMYCIN C)

The objective of this research has been to study the molecular basis for chromosome aberration formation. Predicated on a variety of data, Mitomycin C (MMC)-induced DNA damage has been postulated to cause the formation of chromatid breaks (and gaps) by preventing the replication of regions of the genome prior to mitosis. The basic protocol for these experiments involved treating synchronized Hela cells in G(,1)-phase with a 1 (mu)g/ml dose of MMC for one hour. After removing the drug, cells were then allowed to progress to mitosis and were harvested for analysis by selective detachment. Utilizing the alkaline elution assay for DNA damage, evidence was obtained to support the conclusion that Hela cells can progress through S-phase into mitosis with intact DNA-DNA interstrand crosslinks. A higher level of crosslinking was observed in those cells remaining in interphase compared to those able to reach mitosis at the time of analysis. Dual radioisotope labeling experiments revealed that, at this dose, these crosslinks were associated to the same extent with both parental and newly replicated DNA. This finding was shown not to be the result of a two-step crosslink formation mechanism in which crosslink levels increase with time after drug treatment. It was also shown not to be an artefact of the double-labeling protocol. Using neutral CsCl density gradient ultracentrifugation of mitotic cells containing BrdU-labeled newly replicated DNA, control cells exhibited one major peak at a heavy/light density. However, MMC-treated cells had this same major peak at the heavy/light density, in addition to another minor peak at a density characteristic for light/light DNA. This was interpreted as indicating either: (1) that some parental DNA had not been replicated in the MMC treated sample or; (2) that a recombination repair mechanism was operational. To distinguish between these two possibilities, flow cytometric DNA fluorescence (i.e., DNA content) measurements of MMC-treated and control cells were made. These studies revealed that the mitotic cells that had been treated with MMC while in G(,1)-phase displayed a 10-20% lower DNA content than untreated control cells when measured under conditions that neutralize chromosome condensation effects (i.e., hypotonic treatment). These measurements were made under conditions in which the binding of the drug, MMC, was shown not to interfere with the stoichiometry of the ethidium bromide-mithramycin stain. At the chromosome level, differential staining techniques were used in an attempt to visualize unreplicated regions of the genome, but staining indicative of large unreplicated regions was not observed. These results are best explained by a recombinogenic mechanism. A model consistent with these results has been proposed.^

GCN5 maintains genome stability during development

The histone acetyltransferase, GCN5, is essential for survival of mice during embryogenesis. GCN5 null embryos die early during development due to increased apoptosis. We have demonstrated that the increased apoptosis in associated with increased p53 protein levels. Loss of p53 rescues the embryonic apoptosis in the GCN5 null embryos. These results raised the question of what molecular trigger leads to p53 stabilization and cell death in the absence of GCN5. p53 is generally referred to as the gatekeeper of the cell, monitoring cellular responses to DNA damage, genotoxic stress, and other unfavorable conditions in the cell. Therefore, we examined individual cells in wild type and mutant embryos for gross chromosomal aberrations that might trigger a genome integrity checkpoint. Karyotype analysis indicates that approximately 30% of the cells in an E8.5 GCN5 null embryo display chromosomal aberrations, predominantly chromosomal end adhesions and associations. In wild type E8.5 embryos, only 6% of the cells have chromosomal aberrations. Recent data using telomeric FISH demonstrates that cells from GCN5 null embryos have a decreased telomeric signal. Telomere maintenance is essential for maintaining genome integrity. Telomeric defects are associated with loss of chromosomes and chromosomal rearrangements that can lead to detrimental gene fusions involved in many types of cancers. Little is known about the chromatin structures present near the telomeric ends, or whether any of the telomere-associated proteins are subject to post-translational modification such as acetylation. Our results are the first data to demonstrate the involvement of a histone acetyltransferase, GCN5, in maintaining genome integrity through telomere maintenance and/or capping. ^

B cells in ADA deficiency

Deficiency of the enzyme adenosine deaminase (ADA) results in severe lymphopenia in humans. Mice with an inactivating mutation in the ADA gene also exhibit profound lymphopenia, as well as pulmonary insufficiency and ribcage abnormalities. In fact, the mouse model has a phenotype that is remarkably similar to that of the human disease, making the mice valuable tools for unraveling the mechanism of lymphocyte destruction in absence of this housekeeping gene. T cell deficiency in ADA deficiency has been extensively studied by others, revealing a block in early thymocyte development. In contrast, our studies revealed that early B cell development in the bone marrow is normal. ADA-deficient mice, however, exhibit profound defects in germinal center formation, preventing antigen-dependent B cell maturation in the spleen. ADA-deficient spleen B cells display significant defects in proliferation and activation signaling, and produce more IgM than their normal counterparts, suggesting that extrafollicular plasmablasts are overrepresented. B cells from ADA-deficient mouse spleens undergo apoptosis more readily than those from normal mouse spleens. Levels of ADA's substrates, adenosine and 2′-deoxyadenosine, are elevated in both bone marrow and spleen in ADA-deficient mice. S ′-adenosyihomoeysteine hydrolase (SAH hydrolase) activity is significantly inhibited in both locales, as well. dATP levels, though, are only elevated in spleen, where B cell development is impaired, and not in bone marrow, where B cell ontogeny is normal. This finding points to dATP as the causative agent of lymphocyte death in ADA deficiency. ADA deficiency results in inhibition of the enzyme ribonucleotide reductase, thereby depleting nucleoside pools needed for DNA repair. Another mouse model that lacks a functional gene encoding a protein involved in DNA repair and/or cell cycle checkpoint regulation, p53-binding protein 1, exhibits blocks in T and B cell development that are similar to those seen in ADA-deficient mice. Unraveling the mechanisms of lymphocyte destruction in ADA deficiency may further understanding of lymphocyte biology, facilitate better chemotherapeutic treatment for lymphoproliferative diseases, and improve gene and enzyme therapy regimens attempted for ADA deficiency. ^

The Set1 methyltransferase opposes Ipl1 aurora kinase functions in chromosome segregation

A phosphorylation balance governed by Ipl1 Aurora kinase and the Glc7 phosphatase is essential for normal chromosome segregation in S. cerevisiae . Deletion of SET1, a histone K4 methyltransferase, suppresses the temperature sensitive phenotype of ipl1-2, and loss the catalytic activity of Set1 is important for this suppression. SET1 deletion also suppresses chromosome loss in ipl1-2 cells. Deletion of other Set1 complex components suppresses the temperature sensitivity of ipl1-2 as well. In contrast, SET1 deletion is synthetic lethal combined with glc7-127. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. I find that Set1 methylates conserved lysines in a kinetochore protein, Dam1, a key mitotic substrate of Ipl1/Glc7. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. My studies demonstrate that Set1 has important, unexpected functions in mitosis through modulating the phosphorylation balance regulated by Ipl1/Glc7. Moreover, my findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function. ^

Xp95 is phosphorylated within the proline rich domain during Xenopus oocyte maturation

Xp95 is the Xenopus ortholog of a conserved family of scaffold proteins that have in common an N-terminal Bro1 domain and a C-terminal proline rich domain (PRD). The regulation of this protein family is poorly understood. We previously showed that Xp95 undergoes a phosphorylation-dependant gel mobility shift during meiotic maturation of Xenopus oocytes, the only natural biological system in which post-translational modifications of this family has been demonstrated. Here we characterized Xp95 phosphorylation via two approaches. First, we tested a series of Xp95 fragments for the ability to gel-shift during oocyte maturation, and found that a fragment containing amino acids 705-786 is sufficient to cause a gel-shift. This fragment is within the N-terminal region of Xp95's PRD (N-PRD). Second, we purified phosphorylated Xp95 and by mass spectrometry found that a 5080 Da peptide which maps to N-PRD (amino acids 706-756) contains two phosphorylation sites, one of which is T745, within the conserved CIN85 binding motif. By in vitro protein interaction assays, we that T745 is critical for CIN85/Xp95 interaction, and that Xp95 phosphorylation correlates with loss of binding to CIN85. We also show that an Alix fragment (amino acids 604-789) also undergoes a gel-shift during oocyte maturation and during colcemid-induced mitotic arrest of HeLa cells. These findings indicate that Xp95/Alix is phosphorylated on the PRD during M phase induction and that the PRD phosphorylation regulates partner protein interaction. ^