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Abstract Hypertrophic cardiomyopathy (HCM) is a dominant genetic disorder of the myocardium associated with dysfunctional contractile proteins. The major risk of HCM is sudden cardiac death, which may occur even in asymptomatic carriers. Causes are highly heterogeneous. Over 140 different mutations in nine sarcomeric genes have been described to date. The majority of cases (80% or more) may eventually be traced to one of these genes. Although genetic counselling is suggested even if mutations are not known, molecular diagnosis implies new options such as carrier identification or - theoretically - preclinical risk stratification. A scheme according to which cardiologists and clinical and molecular geneticists could cooperate in counselling patients and managing HCM clinically is proposed.

Missense mutations in alpha tropomyosin, cardiac troponin T and B cardiac myosin heavy chain (MHC) cause familial hypertrophic cardiomyopathy (FHC) indicating that FHC affects the sarcomere. A non-missense mutation in cardiac troponin T which is similar to a splice donor mutation functioning as a null allele also causes FHC. Imbalance of sarcomeric proteins can also cause cardiac hypertrophy.

Aims Hypertrophic cardiomyopathy (HCM) is caused by mutations in genes that encode sarcomeric proteins. In this study we investigated the involvement of the sarcomeric myosin binding protein C in the Dutch HCM population.Methods and results We initially screened 22 Dutch index patients for mutations in the MYBPC3 gene, which revealed four different mutations in 14 patients. The 2373insG mutation was identified in 10 apparently unrelated patients. A subsequent screening for the 2373insG mutation in a group of another 237 unrelated HCM patients revealed 50 additional carriers of the same genetic defect. Genotyping with polymorphic repeat markers and intragenic SNPs of the 60 Dutch as well as two German and five North American 2373insG carriers indicated they all share the same haplotype.Conclusion The 2373insG mutation accounts for almost one-fourth of all HCM cases in the Netherlands (60/259), which is predominantly present in the northwestern part of the country (22/66) and is a founder mutation probably originating from the Netherlands. [ABSTRACT FROM PUBLISHER]

DNA topoisomerases (or nicking-closing enzymes) introduce transient swivels into DNA. We studied the reaction of the calf thymus topoisomerase with single-stranded fd DNA. The nicking reaction of this enzyme is accompanied by strong attachment of the enzyme to DNA. We report conditions under which the intermediate complexes between DNA and topoisomerase are formed and describe some of their properties. For the analyses a filter-binding assay was used which is based on the adsorption of DNA-protein associates to Whatman glass-fiber filters [see Coombs and Pearson (1978) Proc. Natl Acad. Sci. U.S.A. 75, 5291-5295]. The rate of appearance of DNAtopoisomerase complexes on the filter is a function of the enzyme concentration in the assay. The equivalent of about 9 × 107 fd DNA molecules/s are found complexed, if the ratio of enzyme molecules to DNA molecules is about 50. The bond between topoisomerase and DNA is stable in 100 mM KOH or 100 mM HCl and resists treatment with 4 M guanidinium hydrochloride, 1 M potassium phosphate (pH 6.8) or phenol. These results indicate a covalent bond between DNA and the enzyme. Furthermore, the calf thymus enzyme attaches to the 3' terminus at the nick. The 5' terminus is dephosphorylated. Filter binding of single-stranded fd DNA as well as relaxation of superhelical PM2 DNA is selectively inhibited by poly(dG) and poly(dG) · poly(dC), but not by other polydeoxynucleotides. This suggests a preference of the enzyme for binding and/or cleaving at dG or dG-rich clusters in the DNA. [ABSTRACT FROM AUTHOR]

Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeSO2OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, recA, recB, recC and rep did not inhibit this excision repair. Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization. 1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RF I) of phage ΦX 174 into the nicked circular form. 2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5′→3′ exonucleolytic activity ofDNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5′→3′ exonucleolytic activity of DNA polymerase I. 3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds. [ABSTRACT FROM AUTHOR]

A protein, called relaxation protein because of its ability to remove superhelical turns in closed-circular DNA, has been isolated and partially characterized from the nuclei of LA9 mouse and HeLa cells. The purification was facilitated by an assay method, with PM2 DNA, which used the fluorescence enhancement of the intercalating dye ethidium bromide upon binding to the closedcircular DNA. The amount of dye bound depends upon the degree of the superhelix density of the DNA. The relaxation products were analysed by the buoyant separation method in CsCl containing ethidium bromide and were shown to be completely relaxed. The purification resulted in a single band in a dodecylsulfate gel electrophoresis with an apparent molecular weight of 37000. The pH optimum is 7.0 and the optimal salt concentration is 0.2 M NaCl. The relaxation protein removes negative as well as positive supercoils, the latter generated by the interaction of ethidium bromide with closed-circular DNA. Relaxation of positive supercoils results, after removal of the dye, in the formation of molecules with superhelix densities exceeding that of native PM2 DNA (0.054). The highest negative superhelix density observed was −0.098 ± 0.001. The corresponding positive superhelix density has been calculated to be + 0.023. A nicking—swivelling—closing mechanism is postulated, but nicked intermediates have so far not been demonstrated. The relaxation protein is not inhibited by known mammahan endonuclease I inhibitors, except for denatured DNA, and does not possess a conventional polynucleotide ligase activity. The relaxation activity was found to be predominantly in the nuclei, with only small amounts present in the cytoplasm and mitochondria. The biological function of transient swivels induced by the relaxation protein is not known. However, transient swivels are considered necessary or useful in the replication of closed-circular DNA or long linear DNA, respectively. Relaxation protein could replace the combined action of an endonuclease and a ligase ahead of the replication fork. Alternatively, transient swivels could be involved in the transcription process. [ABSTRACT FROM AUTHOR]

Reports on a human myosin heavy-chain gene which was shown to code for a cardiac myosin heavy chain of the beta-type. Location of the 5' end of the 14,200-base-pair genomic DNA clone in the head region of the myosin chain; Transcription of the beta-myosin heavy-chain gene in human heart muscle as demonstrated by the S1 nuclease protection technique.

Calf thymus DNA topoisomerase I, which belongs to the eukaryotic type I topoisomerases, is in a typical preparation purified as a set of five major polypeptides with Mr between 70000 and 100000. At least four of these proteins have binding affinity for DNA as was shown by incubating them with radioactive single-stranded DNA after separation in dodecylsulfate polyacrylamide gels and blotting onto nitrocellulose filters. That these polypeptides have DNA relaxing activity was directly demonstrated with protein extracted from single bands of dodecylsulfate/polyacrylamide gels. We consider the 100000 - Mr protein to be the native enzyme. The smaller components are catalytically active fragments of the native topoisomerase most probably arising from limited proteolysis either within the nucleus or during the purification of the enzyme. In two- dimensional non-equilibrium pH-gradient electrophoresis gels the topoisomerase size variants exhibit apparent pI values between 8.1 and 8.3, with small but distinct differences between the components. The calf thymus topoisomerase I, upon binding to phage fd-DNA, protects a stretch of 15–25 nucleotides againt digestion with DNase I. [ABSTRACT FROM AUTHOR]

A slight DNA topoisomerase I activity was detected in highly purified poly(ADP-Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP-ribosylation) reaction occurs in the presence of NAD. Purified topoisomerase I from calf thymus was shown to be ADP-ribosylated by poly(ADP-Rib) polymerase purified from the same tissue. Poly(ADP-ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP-ribosylation. The fact that a slight poly(ADP-Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that toposiomerase I activity can be modified by ADP-ribosylation, may suggest a spacial and functional correlation of these two enzymes in chromatin. [ABSTRACT FROM AUTHOR]