Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase.

The cmd1-6 allele contains three mutations that block Ca2+ binding to calmodulin from Saccharomyces cerevisiae. We find that strains containing cmd1-6 lose viability during cell cycle arrest induced by the mating pheromone alpha-factor. The 50% lethal dose (LD50) of alpha-factor for the calmodulin mutant is almost fivefold below the LD50 for a wild-type strain. The calmodulin mutants are not more sensitive to alpha-factor, as measured by activation of a pheromone-responsive reporter gene. Two observations indicate that activation of the Ca2+-calmodulin-dependent protein phosphatase calcineurin contributes to survival of pheromone-induced arrest. First, deletion of the gene encoding the calcineurin regulatory B subunit, CNB1, from a wild-type strain decreases the LD50 of alpha-factor but has no further effect on a cmd1-6 strain. Second, a dominant constitutive calcineurin mutant partially restores the ability of the cmd1-6 strain to survive exposure to alpha-factor. Activation of the Ca2+-calmodulin-dependent protein kinase (CaMK) also contributes to survival, thus revealing a new function for this enzyme. Deletion of the CMK1 and CMK2 genes, which encode CaMK, decreases the LD50 of pheromone compared with that for a wild-type strain but again has no effect in a cmd1-6 strain. Furthermore, the LD50 of alpha-factor for a mutant in which the calcineurin and CaMK genes have been deleted is the same as that for the calmodulin mutant. Finally, the CaMK and calcineurin pathways appear to be independent since the ability of constitutive calcineurin to rescue a cmd1-6 strain is not blocked by deletion of the CaMK genes.

WRN helicase expression in Werner syndrome cell lines.

Mutations in the chromosome 8p WRN gene cause Werner syndrome (WRN), a human autosomal recessive disease that mimics premature aging and is associated with genetic instability and an increased risk of cancer. All of the WRN mutations identified in WRN patients are predicted to truncate the WRN protein with loss of a C-terminal nuclear localization signal. However, many of these truncated proteins would retain WRN helicase and/or nuclease functional domains. We have used a combination of immune blot and immune precipitation assays to quantify WRN protein and its associated 3'-->5' helicase activity in genetically characterized WRN patient cell lines. None of the cell lines from patients harboring four different WRN mutations contained detectable WRN protein or immune-precipitable WRN helicase activity. Cell lines from WRN heterozygous individuals contained reduced amounts of both WRN protein and helicase activity. Quantitative immune blot analyses indicate that both lymphoblastoid cell lines and fibroblasts contain approximately 6 x 10(4)WRN molecules/cell. Our results indicate that most WRN mutations result in functionally equivalent null alleles, that WRN heterozygote effects may result from haploinsufficiency and that successful modeling of WRN pathogenesis in the mouse or in other model systems will require the use of WRN mutations that eliminate WRN protein expression.

Genetic instability and hematologic disease risk in Werner syndrome patients and heterozygotes.

Werner syndrome (WRN) is an uncommon autosomal recessive disease in which progeroid features are associated with genetic instability and an elevated risk of neoplasia. We have used the glycophorin A (GPA) somatic cell mutation assay to analyze genetic instability in vivo in WRN patients and heterozygotes. GPA variant frequencies were determined for 11 WRN patients and for 10 heterozygous family members who collectively carry 10 different WRN mutations. Genetic instability as measured by GPA O/N allele loss variant frequency was significantly increased, and this increase was strongly age-dependent in WRN patients. GPA O/N allele loss variants were also significantly elevated in heterozygous family members, thus providing the first evidence for in vivo genetic instability in heterozygous carriers in an autosomal recessive genetic instability syndrome. Our results and comparable data on other human genetic instability syndromes allow an estimate of the level of genetic instability that increases the risk of human bone marrow dysfunction or neoplasia.

Statistical modelling and phylogenetic analysis of a deaminase domain.

Deamination reactions are catalyzed by a variety of enzymes including those involved in nucleoside/nucleotide metabolism and cytosine to uracil (C-->U) and adenosine to inosine (A-->I) mRNA editing. The active site of the deaminase (DM) domain in these enzymes contains a conserved histidine (or rarely cysteine), two cysteines and a glutamate proposed to act as a proton shuttle during deamination. Here, a statistical model, a hidden Markov model (HMM), of the DM domain has been created which identifies currently known DM domains and suggests new DM domains in viral, bacterial and eucaryotic proteins. However, no DM domains were identified in the currently predicted proteins from the archaeon Methanococcus jannaschii and possible causes for, and a potential means to ameliorate this situation are discussed. In some of the newly identified DM domains, the glutamate is changed to a residue that could not function as a proton shuttle and in one instance (Mus musculus spermatid protein TENR) the cysteines are also changed to lysine and serine. These may be non-competent DM domains able to bind but not act upon their substrate. Phylogenetic analysis using an HMM-generated alignment of DM domains reveals three branches with clear substructure in each branch. The results suggest DM domains that are candidates for yeast, platyhelminth, plant and mammalian C-->U and A-->I mRNA editing enzymes. Some bacterial and eucaryotic DM domains form distinct branches in the phylogenetic tree suggesting the existence of common, novel substrates.

Statistical modeling, phylogenetic analysis and structure prediction of a protein splicing domain common to inteins and hedgehog proteins.

Inteins, introns spliced at the protein level, and the hedgehog family of proteins involved in eucaryotic development both undergo autocatalytic proteolysis. Here, a specific and sensitive hidden Markov model (HMM) of protein splicing domain shared by inteins and the hedgehog proteins has been trained and employed for further analysis. The HMM characterizes the common features of this domain including the position where a site-specific DNA endonuclease domain is inserted in the majority of the inteins. The HMM was used to identify several new putative inteins, such as that in the Methanococcus jannaschii klbA protein, and to generate a multiple sequence alignment of sequences possessing this domain. Phylogenetic analysis suggests that hedgehog proteins evolved from inteins. Secondary and tertiary structure predictions suggest that the domain has a structure similar to a beta-sandwich. Similarities between the serine protease cleavage mechanism and the protein splicing reaction mechanism are discussed. Examination of the locations of inteins indicates that they are not inserted randomly in an extein, but are often inserted at functionally important positions in the host proteins. A specific and sensitive HMM for a domain present in klbA proteins identified several additional bacterial and archaeal family members, and analysis of the site of insertion of the intein suggests residues that may be functionally important. This domain may play a role in formation of surface-associated protein complexes.

The effects of colectomy on immediate-early proto-oncogene expression and hepatic regeneration in the rat.

The intact liver exists in a state of replicative quiescence. The factor(s) responsible for maintaining this state and their tissue sources have yet to be identified. Because the colon synthesizes and/or absorbs numerous agents that inhibit hepatocyte proliferation, the principle purpose of this study was to determine whether total colectomy would result in the conversion of quiescent livers to a state of replicative competence. Thus, adult, male Sprague-Dawley rats (250-300 g) were randomized to undergo either total colectomy with ileostomy or sham surgery. Thereafter, rats were sacrificed (N=3-6/group) at times 15 and 30 min and 1, 2, 6, and 24 hr and the livers analyzed by Northern blot analyses for mRNA of the following immediate-early proto-oncogenes (IEP genes): c-fos, c-jun, and c-myc. Rats sacrificed at 24 hr also had hepatic regenerative activity documented by [3H]thymidine incorporation into hepatic DNA. The results of the study revealed that within 15 min, c-fos and c-jun mRNA expression increased in colectomized rats, with peak expression occurring at 30 and 60 min, respectively. c-myc mRNA expression was more delayed, with peak expression occurring at 6 hr post-colectomy. IEP gene expression also increased somewhat in sham-colectomy controls but the increases were not as prompt and, in general, were of lower magnitude than those in the colectomy group. Despite the differences in IEP gene expression between the two groups, [3H]thymidine incorporation at 24 hr was similar (mean+/-SE: colectomy group, 17.2+/-2.6 dpm/microg DNA; sham-colectomy controls, 14.8+/-1.4 dpm/microg DNA). To determine whether the increases in IEP gene expression expedite or augment the hepatic regenerative response to partial hepatectomy (PHx), rats that had undergone colectomy or sham colectomy 1 hr earlier and rats with no previous abdominal surgery then underwent a 70% PHx and were sacrificed at 8, 16, and 24 hr thereafter. At each time interval, [3H]thymidine incorporation was documented and found to be similar in the three groups. In conclusion, the results of this study indicate that total colectomy, and to a lesser extent abdominal surgery, induces the conversion of an intact, quiescent liver to a state of replicative competence. The results also suggest that, in addition to colectomy, the presence of mitogens and/or co-mitogens is required for further progression of hepatocytes through the cell cycle. Finally, a "primed" liver does not respond more promptly or vigorously to a regenerative stimulus than a "resting" liver.

The Fanconi anemia complementation group A protein contains a peroxidase domain.

Computational analysis of the Fanconi anemia (FA) complementation group A protein suggests that it contains a peroxidase domain. FA proteins may be part of a general mechanism that protects cells from oxidative damage.

Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation.

The essential calmodulin genes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe were precisely replaced with genes encoding fusions between calmodulin and the green fluorescent protein (GFP). In living budding yeast the GFP-calmodulin fusion protein (GFP-Cmd1p) localized simultaneously to sites of cell growth and to the spindle pole body (SPB), the yeast analog of the centrosome. Having demonstrated proper localization of GFP-calmodulin in budding yeast, we examined the localization of a fusion between GFP and calmodulin (GFP-Camlp) in fission yeast, where calmodulin had not been localized by any method. We find GFP-Camlp also localizes both to sites of polarized cell growth and to the fission yeast SPB. The localization of calmodulin to the SPB by GFP fusion was confirmed by indirect immunofluorescence. Antiserum to S. pombe calmodulin labeled the ends of the mitotic spindle stained with anti-tubulin antiserum. This pattern was identical to that seen using antiserum to Sad1p, a known SPB component. We then characterized the defects in a temperature-sensitive S. pombe calmodulin mutant. Mutant cam1-E14 cells synchronized in S phase completed DNA synthesis, but lost viability during transit of mitosis. Severe defects in chromosome segregation, including hypercondensation, fragmentation, and unequal allocation of chromosomal material were observed. Immunofluorescence analysis of tubulin revealed a population of cells containing either broken or mislocalized mitotic spindles, which were never observed in wild-type cells. Taken together with the subcellular localization of calmodulin, the observed spindle and chromosome segregation defects suggest that calmodulin performs an essential role during mitosis at the fission yeast SPB.

WRN mutations in Werner syndrome.

Werner syndrome (WS) is one of a group of human genetic diseases that have recently been linked to deficits in cellular helicase function. We review the spectrum of WS-associated WRN mutations, the organization and potential functions of the WRN protein, and potential mechanistic links between the loss of WRN function and pathogenesis of the WS clinical and cellular phenotypes.

The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains.

Prior sequence analysis studies have suggested that bacterial ribonuclease (RNase) Ds comprise a complete domain that is found also in Homo sapiens polymyositis-scleroderma overlap syndrome 100 kDa autoantigen and Werner syndrome protein. This RNase D 3'-->5' exoribonuclease domain was predicted to have a structure and mechanism of action similar to the 3'-->5' exodeoxyibonuclease (proofreading) domain of DNA polymerases. Here, hidden Markov model (HMM) and phylogenetic studies have been used to identify and characterise other sequences that may possess this exonuclease domain. Results indicate that it is also present in the RNase T family; Borrelia burgdorferi P93 protein, an immunodominant antigen in Lyme disease; bacteriophage T4 dexA and Escherichia coli exonuclease I, processive 3'-->5' exodeoxyribonucleases that degrade single-stranded DNA; Bacillus subtilis dinG, a probable helicase involved in DNA repair and possibly replication, and peptide synthase 1; Saccharomyces cerevisiae Pab1p-dependent poly(A) nuclease PAN2 subunit, required for shortening mRNA poly(A) tails; Caenorhabditis elegans and Mus musculus CAF1, transcription factor CCR4-associated factor 1; Xenopus laevis XPMC2, prevention of mitotic catastrophe in fission yeast; Drosophila melanogaster egalitarian, oocyte specification and axis determination, and exuperantia, establishment of oocyte polarity; H.sapiens HEM45, expressed in tumour cell lines and uterus and regulated by oestrogen; and 31 open reading frames including one in Methanococcus jannaschii . Examination of a multiple sequence alignment and two three-dimensional structures of proofreading domains has allowed definition of the core sequence, structural and functional elements of this exonuclease domain.

Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin.

Eukaryotic chromosome segregation depends on the mitotic spindle apparatus, a bipolar array of microtubules nucleated from centrosomes. Centrosomal microtubule nucleation requires attachment of gamma-tubulin ring complexes to a salt-insoluble centrosomal core, but the factor(s) underlying this attachment remains unknown. In budding yeast, this attachment is provided by the coiled-coil protein Spc110p, which links the yeast gamma-tubulin complex to the core of the yeast centrosome. Here, we show that the large coiled-coil protein kendrin is a human orthologue of Spc110p. We identified kendrin by its C-terminal calmodulin-binding site, which shares homology with the Spc110p calmodulin-binding site. Kendrin localizes specifically to centrosomes throughout the cell cycle. N-terminal regions of kendrin share significant sequence homology with pericentrin, a previously identified murine centrosome component known to interact with gamma-tubulin. In mitotic human breast carcinoma cells containing abundant centrosome-like structures, kendrin is found only at centrosomes associated with spindle microtubules.

Ca2+ binding to calmodulin and its role in Schizosaccharomyces pombe as revealed by mutagenesis and NMR spectroscopy.

As a first step toward identifying the important structural elements of calmodulin from Schizosaccharomyces pombe, we examined the ability of heterologous calmodulins and Ca(2+)-binding site mutant S. pombe calmodulins to replace the essential cam1+ gene. A cDNA encoding vertebrate calmodulin allows growth of S. pombe. However, calmodulin from Saccharomyces cerevisiae does not support growth even though the protein is produced at high levels. With one exception, all mutant S. pombe calmodulins with one or more intact Ca(2+)-binding sites allow growth at 21 degrees C. A mutant containing only an intact Ca(2+)-binding site 3 fails to support growth, as does S. pombe calmodulin with all four Ca(2+)-binding sites mutated. Several of the mutant proteins confer a temperature-sensitive phenotype. Analysis of the degree of temperature sensitivity allows the Ca(2+)-binding sites to be ranked by their ability to support fission yeast proliferation. Site 2 is more important than site 1, which is more important than site 4, which is more important than site 3. A visual colony color screen based on the fission yeast ade1+ gene was developed to perform these genetic analyses. To compare the Ca(2+)-binding properties of individual sites to their functional importance for viability, Ca2+ binding to calmodulin from S. pombe was studied by 1H NMR spectroscopy. NMR analysis indicates a Ca(2+)-binding profile that differs from those previously determined for vertebrate and S. cerevisiae calmodulins. Ca(2+)-binding site 3 has the highest relative affinity for Ca2+, while the affinities of sites 1, 2, and 4 are indistinguishable. A combination of an in vivo functional assay and an in vitro physical assay reveals that the relative affinity of a site for Ca2+ does not predict its functional importance.

Statistical modeling and analysis of the LAGLIDADG family of site-specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the HNH family.

The LAGLIDADG and HNH families of site-specific DNA endonucleases encoded by viruses, bacteriophages as well as archaeal, eucaryotic nuclear and organellar genomes are characterized by the sequence motifs 'LAGLIDADG' and 'HNH', respectively. These endonucleases have been shown to occur in different environments: LAGLIDADG endonucleases are found in inteins, archaeal and group I introns and as free standing open reading frames (ORFs); HNH endonucleases occur in group I and group II introns and as ORFs. Here, statistical models (hidden Markov models, HMMs) that encompass both the conserved motifs and more variable regions of these families have been created and employed to characterize known and potential new family members. A number of new, putative LAGLIDADG and HNH endonucleases have been identified including an intein-encoded HNH sequence. Analysis of an HMM-generated multiple alignment of 130 LAGLIDADG family members and the three-dimensional structure of the I- Cre I endonuclease has enabled definition of the core elements of the repeated domain (approximately 90 residues) that is present in this family of proteins. A conserved negatively charged residue is proposed to be involved in catalysis. Phylogenetic analysis of the two families indicates a lack of exchange of endonucleases between different mobile elements (environments) and between hosts from different phylogenetic kingdoms. However, there does appear to have been considerable exchange of endonuclease domains amongst elements of the same type. Such events are suggested to be important for the formation of elements of new specficity.

Immediate-early protooncogene expression and liver function following various extents of partial hepatectomy in the rat.

Immediate-early protooncogenes (IEP) are thought to play an important role in hepatocyte replication. Whether the extent of their expression correlates with the strength of the proliferative stimulus and subsequent regenerative activity has yet to be documented in vivo. Data are also lacking with respect to the level at which liver disease is associated with biochemical evidence of hepatic dysfunction. Thus, the objectives of this study were to determine whether a correlation exists between IEP gene mRNA expression and varying extents of partial hepatectomy (PHx) and to document the extent of resection required to result in increases in serum bilirubin levels. Eighty-nine adult, male Sprague-Dawley rats underwent either sham surgery or 20%, 35%, 55%, 70% or 90% PHx. Postoperatively, rats were killed (N = 3-6/group) at 15 and 30 mins and 8 and 24 hrs for c-fos, c-jun, and c-myc mRNA expression by northern blot analyses. Rats killed at 24 hrs also had hepatic regenerative activity documented by [3H]thymidine incorporation into hepatic DNA and serum bilirubin determinations. While c-fos mRNA expression at 15 mins and c-myc mRNA expression at 8 hrs after PHx did not correlate with the extent of PHx (r2 = 0.478 and 0.018, respectively), a weak correlation existed between c-jun mRNA expression at 30 mins and the extent of PHx (r2 = 0.662, P < 0.05). In terms of IEP mRNA expression and hepatic regenerative activity, a strong correlation existed between c-fos mRNA expression and [3H]thymidine incorporation (r2 = 0.851, P < 0.01) but not c-jun or c-myc mRNA expression. Compared to sham operated controls, [3H]thymidine incorporation was 2.0x, 3.4x, 3.2x, 7.8x, and 2.2x increased following 20%, 35%, 55%, 70%, and 90% PHx, respectively. Serum bilirubin levels remained unchanged until 70% PHx, when they increased from baseline values of 0.54+/-0.05 mg/dl to 1.02+/-0.15 mg/dl (P < 0.05). A further increase occurred following 90% PHx (1.83+/-0.30 mg/dl, P < 0.01). In conclusion these findings indicate that c-fos mRNA expression 15 mins after PHx correlates with hepatic regenerative activity but not the strength of the regenerative stimulus and that hepatic parenchymal loss of 55-70% must occur prior to the detection of elevated serum bilirubin levels. The results also indicate that relative to a 70% PHx, 90% PHx is associated with decreased rather than increased hepatic regenerative activity.

Cell fusion corrects the 4-nitroquinoline 1-oxide sensitivity of Werner syndrome fibroblast cell lines.

We have shown that Werner syndrome (WRN) fibroblast cell lines are unusually sensitive to the DNA-damaging agent 4-nitroquinoline 1-oxide (4NQO), though not to gamma radiation or to hydrogen peroxide. The fusion of 4NQO-sensitive WRN and 4NQO-resistant control fibroblast cell lines generated proliferating WRN x control cell hybrids that expressed WRN protein and were 4NQO-resistant. These results establish the recessive nature of 4NQO sensitivity in WRN cell lines and provide a cellular assay for WRN protein function.