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Expression of the gene for Batten disease (CLN3) was studied in Escherichia coli and in a cell-free rabbit reticulocyte expression systems. A full-length recombinant fusion CLN3 protein was not produced in the bacterial systems used. However, both N-terminal fragment encompassing 246 amino acids and short C-terminal fragment containing 428-438 amino acids of the CLN3 protein were successfully overexpressed in bacteria. Further studies showed that the C-terminal sequence of the CLN3 protein corresponding to the 356-438 amino acid residues was responsible for inhibition of protein synthesis in bacteria. The full-length CLN3 gene product was readily synthesized in vitro in the cell-free rabbit reticulocyte expression system. The product obtained, corresponding to core CLN3 protein, showed an approximate molecular weight of 43 kDa. Immunoprecipitation of this product with pAb to 4-19 amino acids of the CLN3 protein allows us to suggest that CLN3 protein translation starts at Met-1. Show less

The late infantile and juvenile variants of Batten disease are genetically distinct neurodegenerative disorders. Hallmarks of Batten disease include cognitive and motor decline, seizures and blindness due to retinitis pigmentosa. Recently, the CLN3 gene responsible for the juvenile variant has been cloned. Also, apoptosis was proven to be the mechanism by which neurons and photoreceptors die. This paper provides mechanistic support for the occurrence of apoptosis in this disease: There was marked upregulation of Bcl-2 in brain from the late infantile and juvenile types at the protein and RNA levels both by immunocytochemistry and by Northern blot analysis; there were also a 42% to 197% increase in brain ceramide determinations in brains from three patients with the juvenile type and three patients with the late infantile type. Double immunolabeling of brain sections for apoptosis and Bcl-2 supported a protective role for Bcl-2 in the juvenile form of Batten disease. These results raise the possibility that the intact CLN3 gene is normally antiapoptotic, and that it could be an upstream regulator of ceramide. Show less

The carboxyl terminal of the predicted amino acid sequence of the Batten disease CLN3 gene protein is CQLS. This motif is expected to be a site for farnesylation at the cysteine residue. In order to determine whether this is indeed farnesylated we have carried out the in-vitro prenylation of tetrapeptides CVLS, CAIL and CQLS using a farnesyl transferase preparation from bovine brain. The data shows that the CQLS is a good acceptor of a farnesyl group similar to CVLS while it is a poor acceptor of a geranylgeranyl group unlike CAIL, which is a good acceptor of a geranylgeranyl group. This suggests that the CLN3 gene product may be a farnesylated protein. Show less

The recent identification of the genes and the mutations underlying infantile neuronal ceroid lipofuscinosis and juvenile onset neuronal ceroid lipofuscinosis facilitates specific DNA-based diagnostics for the disorders. We have developed a solid-phase minisequencing test for the identification of the major Finnish INCL mutation, an A to T transversion at nucleotide position 364 of the palmitoyl protein thioesterase gene on chromosome 1. This test has been applied for prenatal diagnosis and for identification of disease carriers in INCL families. For population-based screening for INCL carriers the coverage of the test would be 98%. In addition, by combining the solid-phase minisequencing test with whole genome preamplification, we have developed a procedure that allows reliable identification of the INCLFin-mutation in single blastomeres from in-vitro-fertilized embryos. This method is applicable for preimplantation diagnosis, and thus it offers an alternative to early prenatal diagnosis in the prevention of INCL. A modification of the solid-phase minisequencing test was devised for detection of the major INCL mutation, a 1.02 kb deletion in the CLN3 gene on chromosome 16. The coverage of this test for diagnosis of INCL and identification of carriers is 90% in Finland and > 80% worldwide. Show less

SST2 plays an important role in the sensitivity of yeast cells to pheromone and in recovery from pheromone-induced G1 arrest. Recently, a family of Sst2p homologs that act as GTPase-activating proteins (GAPs) for G alpha subunits has been identified. We have identified an interaction between Sst2p and the previously identified Mpt5p by using the two-hybrid system. Loss of Mpt5p function resulted in a temperature-sensitive growth phenotype, an increase in pheromone sensitivity, and a defect in recovery from pheromone-induced G1 arrest, although the effects on pheromone response and recovery were mild in comparison to those of sst2 mutants. Overexpression of either Sst2p or Mpt5p promoted recovery from G1 arrest. Promotion of recovery by overexpression of Mpt5p required Sst2p, but the effect of overexpression of Sst2p was only partially dependent on Mpt5p. Mpt5p was also found to interact with the mitogen-activated protein kinase homologs Fus3p and Kss1p, and an mpt5 mutation was able to suppress the pheromone arrest and mating defects of a fus3 mutant. Because either mpt5 or cln3 mutations suppressed the fus3 phenotypes, interactions of Mpt5p with the G1 cyclins and Cdc28p were tested. An interaction between Mpt5p and Cdc28p was detected. We discuss these results with respect to a model in which Sst2p plays a role in pheromone sensitivity and recovery that acts through Mpt5p in addition to a role as a G alpha GAP suggested by the analysis of the Sst2p homologs. Show less

Most reported mutations that affect lipoprotein metabolism are found within the coding sequences of genes. Recently, a few mutations that occur within promoter sequences have been detected. These promoter sequence variants are the topic of the present review. Some of these variants are fairly common genomic variants in the promoter regions for candidate genes in lipoprotein metabolism, such as APOA1, APOC3, LPA, and LPL. It is possible that such regulatory sequence variants can result in chronic, modestly altered levels of expression of qualitatively normal gene products. This might have a cumulative effect on quantitative phenotypes, such as plasma lipoprotein concentrations, over the long term. Such an effect might not be detected by existing clinical, biochemical, and/or physiological assays. At present, the most consistent evidence from several lines of experiments indicates that genomic variation in the APOC3 promoter creates slightly elevated plasma triglyceride concentrations within the physiologic range. This altered expression appears to predispose to hypertriglyceridemia in the presence of secondary factors. Genetic variants that produce small effects on promoter function might thus be one component of the predisposition to complex diseases. The aggregate of many small effects may create or contribute to a background of susceptibility that, under appropriate conditions, leads to development of frank dyslipidemia and atherosclerosis. Show less

Within the core histone octamer each histone H4 interacts with each H2A-H2B dimer subunit through two binding surfaces. Tyrosines play a central role in these interactions with H4 tyrosines 72 and 88 contacting one H2A-H2B dimer subunit, and tyrosine 98 contacting the other. To investigate the roles of these interactions in vivo, we made site-directed amino acid substitutions at each of these tyrosine residues. Elimination of either set of interactions is lethal, suggesting that binding of the tetramer to both dimers is essential. Temperature-sensitive mutants were obtained through single amino acid substitutions at each of the tyrosines. The mutants show both strong positive and negative effects on transcription. Positive effects include Spt- and Sin-phenotypes resulting from mutations at each of the three tyrosines. One allele has a strong negative effect on the expression of genes essential for the G1 cell cycle transition. At restrictive temperature, mutant cells fail to express the CLN1, CLN2, SWI4 and SWI6 genes, and have reduced levels of CLN3 mRNA. These results demonstrate the critical role of histone dimer-tetramer interactions in vivo, and define their essential role in the expression of genes regulating G1 cell cycle progression. Show less

We have identified a novel promoter element that confers M/G1-specific transcription in Saccharomyces cerevisiae. This element, which we call an ECB (early cell cycle box), was first identified in the SWI4 promoter, but it is also present in the promoter of a G1 cyclin CLN3, as well as in the promoters of three DNA replication genes: CDC6, CDC47, and CDC46. Transcripts from all five of these genes oscillate during the cell cycle and peak at the M/G1 boundary, as do isolated ECB elements in reporter constructs. The ECB element contains an Mcm1 binding site to which Mcm1 binds in vitro, and an Mcm1-VP16 fusion, which places a constitutive activator on Mcm1-binding sites in vivo, can deregulate ECB-containing promoters. Mcm1 is a transcription factor that is also required for minichromosome maintenance. We provide evidence that the replication defect of mcm1 mutants can be suppressed by ectopic CDC6 transcription. Periodic expression of SWI4 and CLN3 may be important for cell cycle progression, as we find that these genes are both haploinsufficient and rate limiting for G1 progression. We suggest that ECB-regulated gene products play critical roles in promoting the initiation of S-phase, both by regulating CLN1 and CLN2 transcription and as components of the initiation complex on origins of replication. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of cartilage-capped prominences that develop from the growth centers of the long bones. EXT is genetically heterogeneous, with three loci, currently identified on chromosomes 8q24.1, 11p13, and 19q. The EXT1 gene, located on chromosome 8q24.1, has been cloned and is encoded by a 3.4-kb cDNA. Five mutations in the EXT1 gene have been identified--four germ-line mutations, including two unrelated families with the same mutation, and one somatic mutation in a patient with chondrosarcoma. Four of the mutations identified resulted in frameshifts and premature termination codons, while the fifth mutation resulted in a substitution of leucine for arginine. Loss of heterozygosity (LOH) analysis of chondrosarcomas and chondroblastomas revealed multiple LOH events at loci on chromosomes 3q, 8q, 10q, and 19q. One sporadic chondrosarcoma demonstrated LOH for EXT1 and EXT3, while a second underwent LOH for EXT2 and chromosome 10. A third chondrosarcoma underwent LOH for EXT1 and chromosome 3q. These results agree with previous findings that mutations at EXT1 and multiple genetic events that include LOH at other loci may be required for the development of chondrosarcoma. Show less

In budding yeast, stability of the mitotic B-type cyclin Clb2 is tightly cell cycle-regulated. B-type cyclin proteolysis is initiated during anaphase and persists throughout the G1 phase. Cln-Cdc28 kinase activity at START is required to repress B-type cyclin-specific proteolysis. Here, we show that Clb-dependent kinases, when expressed during G1, are also capable of repressing the B-type cyclin proteolysis machinery. Furthermore, we find that inactivation of Cln- and Clb-Cdc28 kinases is sufficient to trigger Clb2 proteolysis and sister-chromatid separation in G2/M phase-arrested cells, where the B-type cyclin-specific proteolysis machinery is normally inactive. Our results suggest that Cln- and Clb-dependent kinases are both capable of repressing B-type cyclin-specific proteolysis and that they are required to maintain the proteolysis machinery in an inactive state in S and G2/M phase-arrested cells. We propose that in yeast, as cells pass through START, Cln-Cdc28-dependent kinases inactivate B-type cyclin proteolysis. As Cln-Cdc28-dependent kinases decline during G2, Clb-Cdc28-dependent kinases take over this role, ensuring that B-type cyclin proteolysis is not activated during S phase and early mitosis. Show less

We have cloned and sequenced the murine homologue of the human EXT1 gene. At the protein level, these genes show almost complete identity as divergence is limited to only 5 amino acid positions that are scattered about the whole sequence. In addition, similarity searches identified a protein from chromosome III of C. elegans that shows significant similarity to the human and murine EXT/Ext genes. Using high resolution backcross mapping, the murine Ext1 was mapped at 26.55 cM between D15Mit143 and D15Mit153 on mouse chromosome 15. Therefore, Ext1 is part of an evolutionarily conserved linkage group including SDC2/Hspg1, TRHR/Trhr, EXT1/Ext1, MYC/Myc, and TG/Tgn. Show less

We recently cloned a cDNA for CLN3, the gene for juvenile-onset neuronal ceroid lipofuscinosis or Batten disease. To resolve the genomic organization we used a cosmid clone containing CLN3 to sequence the entire gene in addition to 1.1 kb 5' of the start of the published CLN3 cDNA and 0.3 kb 3' to the polyadenylation site. CLN3 is organized into at least 15 exons spanning 15 kb and ranging from 47 to 356 bp. The 14 introns vary from 80 to 4227 bp, and all exon/intron junction sequences conform to the GT/AG rule. Numerous repetitive Alu elements are present within the introns and 5'- and 3'-untranslated regions. The 5' region of the CLN3 gene contains several potential transcription regulatory elements but no consensus TATA-1 box was identified. CLN3 is homologous to 27 deposited human ESTs, and sequence comparisons suggest alternative splicing of the gene and the existence of transcribed sequences upstream to the start of the published CLN3 cDNA. Show less

Beyond their critical role in thrombosis, platelets perform important functions in vascular remodeling, inflammation, and wound repair. Many of these functions are executed by molecules expressed by activated platelets. A novel molecule, activated-platelet protein-1 (APP-1), was identified by a monoclonal antibody against activated rabbit platelets. When platelets were stimulated by thrombin, A23187 or ADP, APP-I was expressed on the platelet surface. APP-1 was also detected in whole cell lysates of platelets, but not on the external surfaces of resting platelets. With maximal activation by thrombin, 15 900 +/- 2800 molecules APP-1 were expressed/platelet. A 2.3-kb cDNA fragment containing a partial coding sequence for APP-1 was isolated from a rabbit bone marrow library by expression cloning with the anti-APP-1 monoclonal antibody. When expressed as a recombinant fusion protein in bacteria, APP-1 bound specifically to poly(A)-Sepharose. The full-length cDNA coding for human APP-1, obtained by DNA hybridization techniques, showed 98.7% amino acid sequence identity with the rabbit protein. Northern analysis with human APP-1 identified a 3.7-kb mRNA transcript in megakaryocytic lines that express transcripts for platelet proteins. Human APP-1 has four ribonucleotide binding domains with ribonucleoprotein 1 and 2 motifs. By virtue of its ribonucleotide binding domains, APP-1 is structurally related to polyadenylate-binding protein, which regulates translation initiation and polyadenylate shortening, and to nucleolysin, a specific effector molecule found in the granules of cytotoxic T lymphocytes. Show less

Genes for the snRNP proteins U1-70K, U1-A, Sm-B'/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3' poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein. Show less

We hypothesized that common genomic variants would be associated with variation in lipoprotein phenotypes in young subjects. We determined genotypes of FABP2, PON, APOC3, and APOE in 188 aboriginal Canadians, aged 9 to 17 years. We found that 13 of 32 possible genotype-phenotype associations were significant: (1) the FABP2 codon 54 genotype was associated with variation in plasma triglycerides (P = .045); (2) the PON codon 192 genotype was associated with variation in plasma total and LDL cholesterol and apoB (P = .0099, P = .0088, and P = .016, respectively); (3) the APOC3 insulin-response-element genotype was associated with variation in plasma triglycerides, HDL cholesterol, apoA-I, the total cholesterol to HDL cholesterol ratio, and the apoB to apoA-I ratio (P = .0014, P = .0069, P = .045, P = .0021, and P = .0081, respectively); and (4) the APOE restriction isotype was associated with variation in plasma LDL cholesterol, apoB, the total cholesterol to HDL cholesterol ratio, and the apoB to apoA-I ratio (P = .025, P = .034, P = .045, and P = .047, respectively). The average young age and relative absence of age-dependent secondary environmental factors could have eased the identification of small genetic effects on lipoprotein phenotypes in this study sample. Show less

Mitogen-activated protein (MAP) kinase phosphatases constitute a growing family of dual specificity phosphatases thought to play a role in the dephosphorylation and inactivation of MAP kinases and are therefore likely to be important in the regulation of diverse cellular processes such as proliferation, differentiation, and apoptosis. For this reason it has been suggested that MAP kinase phosphatases may be tumor suppressors. We have determined the chromosomal locations of three human dual specificity phosphatase genes by fluorescence in situ hybridization and radiation hybrid mapping. The genes were localized to three different chromosomes, MKP2 (DUSP4) to 8p11-p12, MKP3 (DUSP6) to 12q22-q23, and MKPX (DUSP7) to 3p21. This will allow the potential roles of these genes in disease processes to be evaluated. Show less

A total of 36 patients with Batten disease (juvenile-onset neuronal ceroid lipofuscinosis), homozygous or heterozygous for the major mutation, a 1.02-kb deletion, in the CLN3 gene, were studied to relate their genotype to their clinical phenotype. The onset of visual failure and epilepsy was highly concordant in both groups. Great inter- and intrafamilial heterogeneity was demonstrated in the development of mental and physical handicap and in magnetic resonance imaging findings among both homozygous and heterozygous patients. The 1.02-kb deletion in homozygous form was always associated with mental and physical handicap, whereas the heterozygous phenotype could be extremely benign without affecting the intellectual level of the patient. Our data suggest that genetic background, modifying genes, and environmental factors all influence the final phenotype of Batten disease. Show less

The plasma lipid response to changes in dietary fat and cholesterol can vary between individuals. The SstI polymorphism, arising from a cytosine to guanosine substitution in the 3' untranslated region of the APOC3 gene distinguishes between two alleles--S1 and S2. The S2 allele has been associated with elevated plasma triacylglycerol, cholesterol, and apolipoprotein (apo) C-III concentrations. In 90 young men we examined the effect of the same mutation on the response of low-density-lipoprotein (LDL) cholesterol to dietary monounsaturated fat. The frequency for the S2 allele was 0.14. Subjects were fed a low-fat diet for 25 d, followed by a diet rich in monounsaturated fatty acid (22% MUFA, 38% total fat) for 28 d; lipoproteins were measured at the end of each diet. There were no significant differences in initial total cholesterol between subjects with the APOC3*S1/APOC3*S1 (S1/S1) and APOC3*S1/APOC3*S2 (S1/S2) genotypes. After consumption of the diet high in MUFA, significant increases in LDL cholesterol (0.13 mmol/L, P < 0.027) were noted in the S1/S1 subjects whereas a significant decrease was observed in the S1/S2 subjects (-0.18 mmol/L, P < 0.046). Significant genotypic effects were seen for diet-induced changes in LDL cholesterol (P < 0.00034), total cholesterol (P < 0.009), and apo B (P < 0.0014). A study of the effect of the interaction between this mutation with that present in position -76 of the APOA1 gene promoter region (G/A) revealed that both mutations had an additive effect on changes in total cholesterol, LDL cholesterol, and apo B induced by diets. Plasma LDL-cholesterol responsiveness to the diet may be explained, at least in part, by variation at the APOC3 gene locus. Show less

The Saccharomyces cerevisiae gene BTN1, encodes a 408 amino acid putative integral membrane protein, which is 39% identical and 59% similar to the human Cln3p, whose mutant forms are responsible for Batten's disease and for a diminished degradation of mitochondrial ATPase synthase subunit c. Disruption experiments established that Btn1p is not essential for viability, mitochondrial function, or degradation of mitochondrial ATP synthase in yeast. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by multiple cartilage-capped outgrowths from the epiphyses of long bones. In some cases, these osteochondromas progress to malignant chondrosarcomas. Alterations in at least three genes (EXT1, EXT2, and EXT3) can cause this disorder. Two of these have been isolated (EXT1 and EXT2) and encode related members of a putative tumor suppressor family. We report here the genomic structure of the human EXT2 gene consisting of 14 exons (plus 2 alternative exons) covering an estimated 108 kb of chromosome 11p11-13. We have derived the DNA sequences at all exon/intron boundaries throughout this gene-information that is important for the detailed study of mutations in EXT2. We have also characterized the mouse EXT2 cDNA and have mapped the mouse locus to chromosome 2 between D2Mit15 and Pax6. This mouse homolog should enable transgenic knockout experiments to be initiated to further elucidate gene function. Interestingly, sequence comparisons reveal that the human and mouse EXT genes have at least two homologs in the invertebrate Caenorhabditis elegans, indicating that they do not function exclusively as regulators of bone growth. This observation opens the way for a functional analysis of these genes in nematodes and other lower organisms. Show less

Wild-type cells of the budding yeast Saccharbmyces cerevisiae arrest in G1 upon nutrient exhaustion. Cell cycle arrest requires the WHI2 gene since whi2 mutants continue to divide and become abnormally small as nutrients are depleted. Here we show that CLN1 and CLN2 transcript levels in a whi2 strain are higher during exponential growth, and persist longer upon starvation, than in an isogenic wild-type strain. In contrast to CLN1 and CLN2, CLN3 levels declined only at very high cell density and were unaffected by the whi2 mutation. Elevated CLN expression is sufficient to explain the whi2 phenotype since ectopic expression of CLN1 in a nutrient-depleted culture caused cells to continue dividing and interfered with the acquisition of heat resistance. These observations show that, either directly or indirectly, Whi2 negatively regulates G1 cyclin expression. Interestingly extremely high levels of Cln1 induced filamentous growth upon nutrient deprivation, suggesting a direct connection between G1 cyclin activity and morphological responses to poor nutrient conditions. Show less

To describe the variation of the phenotype within families with several individuals with Bardet-Biedl syndrome. The phenotypes of affected siblings in 11 Scandinavian families with two or more members who had at least three of the features: retinal dystrophy, polydactyly, obesity, hypogenitalism, and mental retardation, were compared [corrected]. Individuals without retinal dystrophy were excluded. Intrafamilial variation of expressivity of the features obesity, polydactyly, abnormal radiograms of the extremities, hypogenitalism, short stature, paraplegia, and dental abnormalities was found. The retinal dystrophy varied with respect to both the onset of symptoms and the course of the disease. The morphology of the fundus, however, was consistent within the families. The disorder showed statistically significant genetic linkage to the BBS4 locus on chromosome 15 in the affected siblings in two of the families, but the clinical features in these patients did not differ from the other cases of Bardet-Biedl syndrome. Comparison of siblings with the Bardet-Biedl syndrome showed variation of the typical features. In addition, the course of retinal dystrophy varied. No distinctive clinical features were found to separate the BBS4 phenotype from the remaining patients. Show less

In budding yeast, entry into the mitotic cell cycle, or Start, requires the Cdc28 cyclin-dependent kinase (Cdk) and one of its three associated G1 cyclins, Cln1, Cln2, or Cln3. In addition, two other G1 cyclins, Pcl1 and Pcl2, associate with a second Cdk, Pho85, to contribute to Start. Although Pho85 is not essential for viability, Pcl1,2-Pho85 kinase complexes become essential for Start in the absence of Cln1,2-Cdc28 kinases. In addition, Pho85 interacts with a third cyclin, Pho80, to regulate acid phosphatase gene expression. Other cellular roles for Pho85 cyclin-Cdk complexes are suggested by the multiple phenotypes associated with deletion of PHO85, in addition to Start defects and deregulated acid phosphatase gene expression. Strains with pho80, pcl1, and pcl2 deletions show only a subset of the pho85 mutant phenotypes, suggesting the existence of additional Pho85 cyclins (Pcls). We used two-hybrid screening and database searching to identify seven additional cyclin-related genes that may interact with Pho85. We found that all of the new genes encode proteins that interacted with Pho85 in an affinity chromatography assay. One of these genes, CLG1, was previously suggested to encode a cyclin, based on the protein's sequence homology to Pcl1 and Pcl2. We have named the other genes PCL5, PCL6, PCL7, PCL8, PCL9, and PCL10. On the basis of sequence similarities, the PCLs can be divided into two subfamilies: the Pcl1,2-like subfamily and the Pho80-like subfamily. We found that deletion of members of the Pcl1,2 class of genes resulted in pronounced morphological abnormalities. In addition, we found that expression of one member of the Pcl1,2 subfamily, PCL9, is cell cycle regulated and is decreased in cells arrested in G1 by pheromone treatment. Our studies suggest that Pho85 associates with multiple cyclins and that subsets of cyclins may direct Pho85 to perform distinct roles in cell growth and division. Show less

Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. The product of the Fused locus, Axin, displays similarities to RGS (Regulators of G-Protein Signaling) and Dishevelled proteins. Mutant Fused alleles that cause axial duplications disrupt the major mRNA, suggesting that Axin negatively regulates the response to an axis-inducing signal. Injection of Axin mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signaling through the Wnt pathway. Furthermore, ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in mammals and amphibians. Show less

By controlled addition of galactose to synchronized galactose-limited Saccharomyces cerevisiae cultures, the growth rate could be regulated while external conditions were kept constant. By using this method, the G1 phase duration was modulated and expression of cell cycle-regulated genes was investigated. The expression of the cyclin genes CLN1 and CLN2 was always induced just before bud emergence, indicating that this event marks the decision to pass Start. Thus, G1 phase elongation was not due to a slower accumulation of the CLN1 and CLN2 mRNA levels. Only small differences in CLN3 expression levels were observed. The maximal SWI4 expression preceded maximal CLN1 and CLN2 expression under all conditions, as expected for a transcriptional activator. But whereas SWI4 was expressed at about 10 to 20 min, before CLN1 and CLN2 expression at high growth rates, this time increased to about 300 min below a particular consumption rate at which the G1 phase strongly elongated. In the slower-growing cultures, also an increase in SWI6 expression was observed in the G1 phase. The increase in G1 phase duration below a particular consumption rate was accompanied by a strong increase in the reserve carbohydrate levels. These carbohydrates were metabolized again before bud emergence, indicating that below this consumption rate, a transient increase in ATP flux is required for progression through the cell cycle. Since Start occurred at different cell sizes under different growth conditions, it is not just a certain cell size that triggers passage through Start. Show less

Multiple exostoses is a polygenic disease of bone formation and development characterized by the presence of cartilage-capped osseous projections emanating from the end of the long bones. Two members of a recently defined multigene family of proteins (EXT1 and 2) were shown to be involved in this disease. To investigate the evolutionary relatedness of EXT genes across species we isolated the mouse EXT2 cDNA. As in the human counterpart, the mouse EXT2 cDNA contains an open reading frame of 2154 bp encoding a predicted protein of 718 amino acids. The nucleic acid sequence is 87% identical to the human EXT2 transcript, resulting in an amino acid sequence which is 95% identical to the human protein. The mouse EXT2 gene also shows significant sequence similarity to the mouse and human EXT1 gene. Northern blot analysis shows that this gene is expressed in early stages of embryonic development, and in situ hybridizations suggest that EXT2 plays a role in limb development. The identification of the mouse EXT2 gene will allow functional analysis through insertional inactivation and reverse genetics in mice in order to better understand the formation of exostoses during bone formation. Show less

U1C is one of the three human U1 small nuclear ribonucleoprotein (snRNP)-specific proteins and is important for efficient complex formation between U1 snRNP and the pre-mRNA 5' splice site. We identified a hypothetical open reading frame in Saccharomyces cerevisiae as the yeast homolog of the human U1C protein. The gene is essential, and its product, YU1C, is associated with U1 snRNP. YU1C depletion gives rise to normal levels of U1 snRNP and does not have any detectable effect on U1 snRNP assembly. YU1C depletion and YU1C ts mutants affect pre-mRNA splicing in vivo, and extracts from these strains form low levels of commitment complexes and spliceosomes in vitro. These experiments indicate a role for YU1C in snRNP function. Structure probing with RNases shows that only the U1 snRNA 5' arm is hypersensitive to RNase I digestion when YU1C is depleted. Similar results were obtained with YU1C ts mutants, indicating that U1C contributes to a proper 5' arm structure prior to its base pairing interaction with the pre-mRNA 5' splice site. Show less

The childhood neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurodegenerative disorders characterised by progressive visual failure, neurodegeneration, epilepsy and the accumulation of an autofluorescent lipopigment in neurones and other cells. Three main subtypes have been identified according to age of onset, clinical features and ultrastructural morphology. These are infantile NCL (INCL; CLN1), classical late infantile NCL (LINCL; CLN2) and juvenile NCL (JNCL; CLN3). Several atypical forms of late infantile NCL (LINCL) have also been described including a Finnish variant LINCL (CLN5). The CLN2 gene has been excluded from the CLN1, CLN3 and CLN5 loci. A genome search was initiated using a homozygosity mapping strategy in five classical LINCL and two variant LINCL consanguineous families. A common region of homozygosity was identified on chromosome 11p15 in two of the classical families. Analysis of a further 33 classical LINCL families supported linkage in this region (Zmax = 3.07 at theta = 0.06 at D11S1338). A common region of homozygosity was also observed on chromosome 15q21-23 in the two variant LINCL families. Extension of the analysis to include a further seven families of identical ultrastructural phenotype established linkage to this region (Zmax = 6.00 at theta = 0.00 at D15S1020). Show less

We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function. Show less