Novel mutations at carboxyl terminus of CIC-1 channel in myotonia congenita.

OBJECTIVES: Myotonia congenita (MC), caused by mutations in the muscle chloride channel (CLCN1) gene, can be inherited dominantly or recessively. The mutations at the carboxyl terminus of the CLCN1 gene have been identified in MC patients, but the functional implication of these mutations is unknown. MATERIAL AND METHODS: Direct sequencing of polymerase chain reaction products covering the whole coding region of the CLCN1 gene was performed in a MC family. This study was designed to investigate the clinical manifestations and genetic analysis of the CLCN1 gene. RESULTS: We identified two novel mutations, 2330delG and 1892C>T, from a genetic screening of the CLCN1 gene in the MC family. The 2330delG mutant allele producing a fs793X truncated protein was identified in a heterozygous state in all the patients. The 1892C>T nucleotide change induced a missense mutation (T631I) found in several asymptomatic individuals, indicating that it may not be associated with MC. Intriguingly, the 2330delG mutation was also found in an asymptomatic subject who also carried the 1892C>T mutation. CONCLUSION: The data indicate that the fs793X mutant protein causes dominantly inherited MC. Because the mutation has been found in a recessive pedigree, the fs793X mutation may have a dual inheritance pattern.

Karyotypic evolution of a novel cervid satellite DNA family isolated by microdissection from the Indian muntjac Y-chromosome.

A minilibrary was constructed from DOP-PCR products using microdissected Y-chromosomes of Indian muntjac as DNA templates. Two microclones designated as IM-Y4-52 and IM-Y5-7 were obtained from negative screening of all three cervid satellite DNAs (satellites I, II, and IV). These two microclones were 295 and 382 bp in size, respectively, and shared approximately 70% sequence homology. Southern blot analysis showed that the IM-Y4-52 clone was repetitive in nature with an approximately 0.32-kb register in HaeIII digest. Sequence comparison revealed no similarities to DNA sequences deposited in the GenBank database, suggesting that the microclone sequences were from a novel satellite DNA family designated as cervid satellite V. A subclone of an Indian muntjac BAC clone which screened positive for IM-Y4-52 had a 3,325-bp insert containing six intact monomers, four deleted monomers, and two partial monomers. The consensus sequence of the monomer was 328 bp in length and shared more than 80% sequence homology with every intact monomer. A zoo blot study using IM-Y4-52 as a probe showed that the strong hybridization with EcoRI digested male genomic DNA of Indian muntjac, Formosan muntjac, Chinese muntjac, sambar deer, and Chinese water deer. Female genomic DNA of Indian muntjac, Chinese water deer, and Formosan muntjac also showed positive hybridization patterns. Satellite V was found to specifically localize to the Y heterochromatin region of the muntjacs, sambar deer, and Chinese water deer and to chromosome 3 of Indian muntjac and the X-chromosome of Chinese water deer.

Epidemiological and genetic studies of myotonic dystrophy type 1 in Taiwan.

To investigate the prevalence and genetic characteristics of myotonic dystrophy type 1 (DM1) in Taiwan, DM-suspected patients and their families identified during the period of 1990-2001 had their clinical records reevaluated and the CTG repeat sizes at the DM1 locus examined. A total of 96 subjects belonging to 26 families were identified as DM1 patients, which gave a minimal disease prevalence of 0.46/100,000 inhabitants. Clinical anticipation was frequently observed in affected families, even in some parent-child pairs with transmission contraction of the CTG repeat size. The inverse correlation between age at onset and CTG repeat length was significant only in patients with small expansions. In addition, a DM1 carrier with a childhood-onset son was found to have CTG length heterogeneity in the range of 40-50, indicating that premutation alleles could be unstable during gametogenesis as well as in somatic tissues. Our data demonstrated that DM1 is a rare disease in Taiwan and showed that transmission contraction of repeat size is more likely to occur in alleles with large repeats.

Haplotype analysis of the myotonic dystrophy type 1 (DM1) locus in Taiwan: implications for low prevalence and founder mutations of Taiwanese myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder caused by a CTG trinucleotide expansion at the DM1 locus. In this study, we investigated the frequency distribution of various CTG repeats in normal alleles and haplotyped the normal and expanded DM1 locus in a group of Taiwanese people. In the 496 normal chromosomes examined, up to 18 alleles with different CTG lengths from 5 to 30 repeats were found and the frequency of (CTG)(>18) alleles was only 1.4% (7/496), predicting a low prevalence of DM1. In addition, there is no absolute association between (CTG)(5-19) alleles and Alu insertion/deletion polymorphism observed on normal chromosomes. All DM1 alleles examined, however, were found to be associated with the Alu insertion. Further detailed genetic analysis demonstrated that at least eight haplotypes, including a new haplotype (L), were present in the Taiwanese population and that all DM1 alleles were with the same haplotype (haplotype A) as that identified in Canadian and Japanese DM1 populations. These findings support the notion that the out-of-Africa DM1 alleles were originated by stepwise expansion from a pool of large-sized normal chromosomes with haplotype A.

Delineation of CTG repeats and clinical features in a Taiwanese myotonic dystrophy family.

Myotonic dystrophy (DM) is an inherited, autosomal dominant muscular disease which is primarily caused by a CTG trinucleotide expansion mutation on chromosome 19q13.3. The size of this trinucleotide repeat is related both to the age of onset and to the severity of the clinical manifestation. This disease is very rare in Taiwan, and clinical and genetic study on DM has not yet been documented in this area. Here, we present both clinical features and degrees of CTG expansion for a Taiwanese DM family. All of the DM patients examined in this family showed obvious clinical manifestations by age 30, which included facial and limb muscle weakness with atrophy, myotonia, and ptosis. In addition, individual DM members also exhibited variable phenotypes, which may reflect the complexity of the pathogenic mechanism. Because the collection of blood specimens was considered to be an invasive procedure, a genetic study on this DM family was performed using buccal cells. Our results confirmed that four members showing classic symptoms of DM had CTG repeat expansion in the DMI locus, and that one member with ptosis and minor muscle weakness in the right foot was a normal homozygote for CTG repeat. These data demonstrate that buccal cells can provide clear and reliable results, and thus, are suitable for a family study of DM.

Identification of the spinocerebellar ataxia type 7 mutation in Taiwan: application of PCR-based Southern blot.

Spinocerebellar ataxia (SCA) type 7 is an autosomal dominant disorder characterized by neural loss, mainly in the cerebellum and regions of the brainstem and particularly the inferior olivary complex. This neurodegeneration disease is associated with expansion of unstable CAG repeats within the 5'-translated region of the SCA7 gene, located on chromosome 3p. We conducted a local survey of the normal population and candidate patients for the analysis of the CAG repeats in the SCA7 gene. The distributions of the CAG repeat units of SCA7 gene in the normal population in Taiwan were established in this study by using the radioactive genomic polymerase chain reaction (PCR). The normal range of CAG repeats is from 6 to 17 repeats, with the more common being around 8-13 repeats. The range is narrower than that reported for other ethnic groups (7-35 CAGs). Meanwhile, by the use of a combination of PCR and Southern blot analysis, one SCA7 family was identified and is reported here. A marked instability of the CAG repeat number during transmission from father to son (41 vs. 100) was observed in the SCA7 family. Clinical anticipation is significant in this family including an infantile case, who was found to have nystagmus from the age of 1 month. To date, the SCA7 mutation has been detected in one of 73 families with autosomal dominant cerebellar ataxia phenotypes, which is about 1.4% of the ataxia families referred to us, compared to 1.4% SCA1, 9.6% SCA2, and 27.3% SCA3/Machado-Joseph disease in our collection. In addition, we demonstrate that the PCR-based Southern blot analysis, with the advantages of sensitivity of PCR and specificity of Southern blot, is a reliable diagnostic method for SCA7 mutation screening. The molecular analysis technique makes possible the quick and accurate diagnosis of SCA7 patients and in the future will hopefully be applied to prenatal screening for SCA7 families.

Application of FTA sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based Southern blotting.

Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7deaza-dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM.

Coupled transcriptional and translational control of cyclin-dependent kinase inhibitor p18INK4c expression during myogenesis.

Terminal differentiation of many cell types involves permanent withdrawal from the cell division cycle. The p18INK4c protein, a member of the p16/INK4 cyclin-dependent kinase (CDK) inhibitor family, is induced more than 50-fold during myogenic differentiation of mouse C2C12 myoblasts to become the predominant CDK inhibitor complexed with CDK4 and CDK6 in terminally differentiated myotubes. We have found that the p18INK4c gene expresses two mRNA transcripts--a 2.4-kb transcript, p18(L), and a 1.2-kb transcript, p18(S). In proliferating C2C12 myoblasts, only the larger p18(L) transcript is expressed from an upstream promoter. As C2C12 cells are induced to differentiate into permanently arrested myotubes, the abundance of the p18(L) transcript decreases. The smaller p18(S) transcript expressed from a downstream promoter becomes detectable by 12 h postinduction and is the predominant transcript expressed in terminally differentiated myotubes. Both transcripts contain coding exons 2 and 3, but p18(L) uniquely contains an additional noncoding 1.2-kb exon, exon 1, corresponding exclusively to the 5' untranslated region (5' UTR). The expression pattern of the shorter p18(S) transcript, but not that of the longer p18(L) transcript, correlates with terminal differentiation of muscle, lung, liver, thymus, and eye lens cells during mouse embryo development. The presence of the long 5' UTR in exon 1 attenuated the translation of p18(L) transcript, while its absence from the shorter p18(S) transcript resulted in significantly more efficient translation of the p18 protein. Our results demonstrate that during terminal muscle cell differentiation, induction of the p18 protein is regulated by promoter switching coupled with translational control.

E2F-mediated growth regulation requires transcription factor cooperation.

Previous studies have indicated that the presence of an E2F site is not sufficient for G1/S phase transcriptional regulation. For example, the E2F sites in the E2F1 promoter are necessary, but not sufficient, to mediate differential promoter activity in G0 and S phase. We have now utilized the E2F1 minimal promoter to test several hypotheses that could account for these observations. To test the hypothesis that G1/S phase regulation is achieved via E2F-mediated repression of a strong promoter, a variety of transactivation domains were brought to the E2F1 minimal promoter. Although many of these factors caused increased promoter activity, growth regulation was not observed, suggesting that a general repression model is incorrect. However, constructs having CCAAT or YY1 sites or certain GC boxes cloned upstream of the E2F1 minimal promoter displayed E2F site-dependent regulation. Further analysis of the promoter activity suggested that E2F requires cooperation with another factor to activate transcription in S phase. However, we found that the requirement for E2F to cooperate with additional factors to achieve growth regulation could be relieved by bringing the E2F1 activation domain to the promoter via a Gal4 DNA binding domain. Our results suggest a model that explains why some, but not all, promoters that contain E2F sites display growth regulation.

Superinduction of mouse epidermal ornithine decarboxylase activity by repeated 12-o-tetradecanoylphorbol-13-acetate treatments.

A correlation of the levels of epidermal protein kinase C (PKC) isozymes, steady state levels of ornithine decarboxylase (ODC) mRNA, and ODC antizyme with the induction of ornithine decarboxylase (ODC) activity by a second repeat 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment to mouse skin was determined. A single application of TPA to female CD-1 mouse skin leads to a dramatic induction of ODC activity (approximately 3 nmol CO2/60 min/mg protein) which peaks at about 5 h after treatment. However, a superinduction of ODC activity (approximately 13 CO2/60 min/mg protein) is observed upon the second TPA application at 48 or 72 h after the first TPA treatment. Prior application of a tumor initiating dose of 7,12-dimethylbenz[a]anthracine to mouse skin did not influence the degree of induction of ODC by a repeat TPA treatment. Western Blot analyses using antibodies specific to PKC alpha, beta, gamma, delta and epsilon indicate detectable levels of PKC alpha, beta, delta and epsilon in mouse epidermal extracts. A time course of the effects of a single topical application of 20 nmol of TPA to the mouse skin indicate that none of PKC isozymes (alpha, beta, gamma, delta and epsilon) were completely downregulated at times (72 h) when ODC was overinduced by TPA. TPA-induced steady state levels of ODC mRNA did not correlate with the degree of superinduction of ODC activity by TPA. The second TPA treatment, 72 h after the first TPA treatment, which leads to superinduction of ODC activity did not decrease the levels of the ODC-antizyme. The results indicate that superinduction of mouse epidermal ODC activity is regulated in part post-transcriptionally and may not be the result of either a loss of PKC isoform(s) or a decrease in the levels of ODC antizyme.

Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter.

To prepare for the DNA synthesis (S) phase of the cell cycle, transcription of many genes required for nucleotide biosynthesis increases. The promoters of several of these genes contain binding sites for the E2F family of transcription factors, and, in many cases, mutation of these sites abolishes growth-regulated transcription. The RNA levels of one family member, E2F1, increase about 15-fold at the G1/S-phase boundary and expression of E2F1 in quiescent cells activates transcription from some G1/S-phase-specific promoters, suggesting that E2F1 plays a critical role in preparing cells to enter S phase. To elucidate the signal transduction pathway leading to the activation of genes required for DNA synthesis, we are investigating the mechanism by which expression of E2F1 is regulated. To determine whether levels of E2F1 mRNA are controlled by changes in promoter activity, we have cloned and characterized the mouse E2F1 promoter. Sequence analysis revealed two sets of overlapping E2F-binding sites located between -12 and -40 relative to the transcription initiation site. We show that these sites bind cellular E2F and that an E2F1 promoter fragment can be activated up to 100-fold by coexpression of E2F proteins. We also show that the activity of this E2F1 promoter fragment increases approximately 80-fold at the G1/S-phase boundary and that this activation is, in part, regulated by G0-specific repression via the E2F sites. However, the E2F sites are not sufficient to mediate growth-regulated transcriptional activity; our results indicate that multiple DNA elements are required for transcription regulation of the E2F1 promoter at the G1/S-phase boundary.

Evidence that inactive p42 mitogen-activated protein kinase and inactive Rsk exist as a heterodimer in vivo.

Mitogen-activated protein kinases (MAP kinases) are active only when phosphorylated. Here we examine whether the activation of Xenopus p42 MAP kinase might involve changes in its association with other proteins as well as changes in its phosphorylation state. We find that when p42 MAP kinase is phosphorylated and active, it is monomeric, and that when p42 MAP kinase is nonphosphorylated and inactive, about half of it is monomeric and half is a component of a 110-kDa complex. We identify Rsk, an 82-kDa protein kinase that can be phosphorylated and partially activated by p42 MAP kinase, as being specifically associated with inactive p42 MAP kinase. It is possible that the complex of inactive p42 MAP kinase and inactive Rsk acts as a single signal reception particle and that the activation of the two kinases may be better described as a fork in a bifurcating signal transduction pathway than as successive levels in a kinase cascade.

Characterization of D-aminoacylase from Alcaligenes denitrificans DA181.

The D-aminoacylase produced by Alcaligenes denitrificans DA181 was a new type of aminoacylase which had both high stereospecificity and specific activity. The molecular weight and isoelectric point of this enzyme were 58,000 and 4.4, respectively. The apparent Km and kcat values of this enzyme for N-acetyl-D-methionine were estimated to be 0.48 mM and 6.24 x 10(4) min-1, respectively. The optimum temperature was 45 degrees C. The enzyme was stable up to 55 degrees C for 1 hr in the presence of 0.2 mg/ml bovine serum albumin. The enzyme was stable in the pH range of 6.0 to 11.0 with an optimum pH of 7.5. This enzyme contained about 2.1 g atom of zinc per mole of enzyme. Enzyme activity was inhibited by incubation with EDTA. The inhibition by EDTA was fully reversed by Co2+ and partially by Zn2+.

Production and Purification of d-Aminoacylase from Alcaligenes denitrificans and Taxonomic Study of the Strain.

A d-aminoacylase-producing microorganism, strain DA181, isolated from soil was identified as Alcaligenes denitrificans subsp. denitrificans. This strain produced about 29,300 units (micromoles of product formed per hour) of d-aminoacylase and 2,300 units of l-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-dl-leucine as the carbon source. The d-aminoacylase was purified 345-fold. The specific activity of the purified enzyme was 108,600 units per mg of protein when N-acetyl-d-methionine was used as a substrate. The apparent molecular weight was 58,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-Acetyl-d-methionine was the favored substrate, followed by N-acetyl-d-phenylalanine. This enzyme had a high stereospecificity, and its hydrolysis of N-acetyl-l-amino acids was almost negligible.