Günter Maass

Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene

SummaryThree mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene were discovered in a pancreas-insufficient patient with cystic fibrosis (CF) who displayed an uncommon combination of almost normal chloride concentration in sweat tests and typical symptoms of gastrointestinal and pulmonary disease. The R553Q mutation was found on the maternal ΔF508-CFTR gene. Codon 553 is located within a consensus motif of the ATP-binding cassette transport proteins at a less conserved position. Other members of this protein superfamily contain a glutamine instead of arginine at the homologous position, suggesting a modulating rather than disease-causing role of the R553Q mutation in CFTR. The amplification refractory mutation system did not detect the R553Q mutation in a further 65 normal, 113 ΔF508, and 91 non-ΔF508 CF chromosomes. The index case carried the R553X nonsense mutation on the paternal chromosome. The R553X mutation was present on a further 9 out of 86 German nonΔF508 CF chromosomes linked with the XV2c-KM19Mp6d9-J44-GATT haplotypes 2-2-2-1-1 and 1-1-2-1-2. The location of R553X on separate haplotypes including both alleles of the intragenic GATT repeat suggests an ancient and/or multiple origins of the R553X mutations. The association of the genotype of the CFTR mutation and the clinical phenotype was assessed for the patients carrying the related genotypes ΔF508/ΔF508 (n = 80), ΔF508/R553X (n = 9) and ΔF508-R553Q/R553X (n = 1). In compound heterozygotes, the median chloride concentration in pilocarpine iontophoresis sweat tests was significantly lower than in the ΔF508 homozygotes (P < 0.01). The patient groups were significantly different with respect to the distributions of the centiles for height (P < 0.001) and weight (P < 0.01) as the most sensitive predictors of the course and prognosis in CF. Growth retardation was more pronounced in the compound heterozygotes.

On the catalytic mechanism of EcoRI and EcoRV A detailed proposal based on biochemical results, structural data and molecular modelling

EcoRI and EcoRV have a very similar active site, as is apparent from a comparison of the structures of their respective protein‐DNA complexes. Based on structural and mechanistic data, as well as detailed molecular modelling presented here, a mechanism for the DNA cleavage by these enzymes is suggested in which the attacking water molecule is activated by the phosphate group 3′ to the scissile phosphodiester bond, and in which the leaving group is protonated by a water molecule associated with the essential cofactor, Mg2+. The mechanism proposed may also apply to other nucleases.

Site-directed mutagenesis in the catalytic center of the restriction endonuclease EcoRI ☆

The catalytic center of the restriction endonuclease (ENase) EcoRI is structurally homologous to that of EcoRV, BamHI and PvuII. Each of these ENases contains a short motif of three to four amino acid (aa) residues which are positioned in a similar orientation to the scissile phosphodiester bond. We have mutated these aa (Pro90, Asp91, Glu111 and Lys113) in EcoRI to determine their individual roles in catalysis. The replacement of Asp91 and Lys113, respectively, by conservative mutations (Ala91, Asn91, Ala113, Gln113, His113 and Leu113) resulted in a reduction of binding affinity and complete loss of cleavage activity. Only Lys113-->Arg substitution still allows to cleave DNA, albeit with a rate reduced by at least four orders of magnitude. Lys113 seems to stabilize the structure of the wild-type (wt) ENase since all five ENase variants with mutations at this position show a strongly enhanced tendency to aggregate. The Ala and Gln mutants of Glu111 bind the recognition sequence slightly stronger than wt EcoRI and cleave it with a low, but detectable rate. Only the Glu111-->Lys mutant, in which the charge is reversed, shows neither binding nor cleavage activity. Pro90 is not important for catalysis, because the Ala90 mutant cleaves DNA with an only slightly reduced rate. Under star conditions, however, this mutant is even more active than wt EcoRI. Therefore, the charged aa Asp91, Glu111 and Lys113 are essential for catalytic activity of the EcoRI ENase.(ABSTRACT TRUNCATED AT 250 WORDS)

Complexes of the single-stranded DNA-binding protein from Escherichia coli (Eco SSB) with poly(dT): an investigation of their structure and internal dynamics by means of electron microscopy and NMR

Based on electron microscopy and NMR spectroscopy it is deduced that Eco SSB binds with moderate cooperativity to polynucleotides. Evidence is provided that the protein binds in its tetrameric form to the nucleic acid forming a nucleosome-like structure. NMR-spectroscopic analysis of the complexes shows that the carboxy-terminal region of the Eco SSB maintains a high flexibility even when the protein is immobilized in large protein-protein clusters.

The single-stranded DNA binding protein of Escherichia coli: physicochemical properties and biological functions

Single-stranded DNA binding proteins fulfil important functions in DNA metabolism. They have been shown to be essential for replication, recombination and repair in bacteria and bacteriophages. The best-studied single-stranded DNA binding proteins are the gene32 protein from T4-phage (gp32) (for reviews, cf. Kowalczykowski et al., 1981; Chase and Williams, 1986; Chase, 1984), the gene5 protein from filamentous phages (gp5) (for reviews, cf. Kowalczykowski et al., 1981), and the E. coli single-stranded DNA binding protein.

Asp-59 is not important for the catalytic activity of the restriction endonuclease EcoRI

The amino acid Asp‐59 was proposed to be involved in EcoRI catalyzed DNA cleavage (Cheng et al., EMBO J. 13, 3927‐35, 1994). We have tested this hypothesis by site directed mutagenesis experiments. The four mutants D59A, D59E, D59G, and D59N bind with similar stability to the specific recognition sequence as wild type EcoRI. The D59E mutant cleaves DNA as fast as the wild type enzyme. Specific activities of the other three mutants are five to tenfold lower. Therefore, we conclude that Asp‐59 is not involved in catalysis of the EcoRI restriction endonuclease. Consequences for catalytic mechanisms of EcoRI and other restriction enzymes are discussed.

Marker haplotype association with growth in German cystic fibrosis patients

SummaryIn 84 families with 101 children with cystic fibrosis (CF) and 103 unaffected siblings, the haplotype of CF chromosomes was determined with six restriction fragment length polymorphism (RFLP) markers that span the CF gene locus. Patient groups with different genotypes in the more distant flanking marker loci MET D, MET H, and D7S8 differed significantly from each other with respect to percentile height and weight, and percentage of weight for height. Patients homozygous 1-1 in met D (TaqI) and met H (TaqI) were thin and tall when homozygous 1-1 in J3.11 (MspI), and small when homozygous 2-2 in J3.11. Heterozygosity in 3.11 and met H and homozygosity 1-1 in met D segregated with the most severe growth retardation. In contrast, growth was normal in patients who were heterozygous in met D and/or had an uncommon KM.19/XV-2c haplotype. Most patients with pancreatic sufficiency and/or borderline sweat test values were carrying rare haplotypes on their CF chromosomes. Adult patients clustered in genotype groups with normal height percentile distributions. This association between haplotype and clinical severity of CF in the German population provides evidence for genetic microheterogeneity of the CF locus, either because of the existence of multiple alleles of the CF gene itself and/or because of the existence of closely linked polymorphic genes that control growth and development and hence modulate the clinical course and prognosis of CF.

Chemical modification studies on the tyrosyl-tRNA synthetase/tRNA complex are in agreement with an antico-operative binding scheme

Abstract Chemical modification experiments on the tyrosyl-tRNA synthetase/tRNA t yr system of Bacillus stearothermophilus reported by Bosshard et al. (1975,1978) which have been interpreted in terms of a binding scheme with one binding site that spans two subunits have been re-evaluated by a more rigorous procedure. The results show that the experiments of Bosshard et al. are in agreement with the antico-operative binding model that has been demonstrated for the Escherichia coli system.

The endonucleolytic reaction catalysed by the EcoRI restriction enzyme is inhibited when the enzyme is in excess over substrate

We have studied the cleavage of radioactively labelled double stranded d(GGAATTCC) in single turnover experiments with substrate and enzyme concentration both being in the ~M range. As can be seen in fig. I the reaction rate increases with enzyme concentration until a 1:1 ratio of substrate and enzyme (dimeric form) is reached, when the enzyme concentration is further increased a sharp decline of the reaction rate is observed.

Stability and dynamics of the complexes of the single strand DNA binding protein (E.coli) with single stranded dna

The single strand DNA binding protein (ssB protein) from E. col i is essential for rep l ica t ion . I t binds cooperatively to single stranded DNA and does not bind to double stranded DNA. The protein consists of four iden t i cal subunits each having a molecular weight of 20,000 daltons. We have determined s t a b i l i t y constants and rate constants for the complex formation of the ssB protein with ol igonucleotides of defined composition and length: each of the four subunits of the ssB protein has a binding s i te for o l igonucleotides of 8 9 residues length. The binding si tes are nearly equivalent and independent. The i n t r i n s i c binding constant K for the complex formation between ssB protein and the various ol igonucleotides and the i r ionic strength dependences are given below :