Backbone and Ile-δ1, Leu,Val methyl <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C,chemical shift assignments for <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> lipase

Lipase r27RCL is a 296-residue, 33 kDa monomeric enzyme with high ester hydrolysis activity, which has significant applications in the baking, paper and leather industries. The lipase gene proRCL from Rhizopus microsporus var. chinensis (also Rhizopus chinensis) CCTCC M201021 was cloned as a fusion construct C-terminal to a maltose-binding protein (MBP) tag, and expressed as MBP-proRCL in an Escherichia coli BL21 trxB (DE3) expression system with uniform ²H,¹³C,¹⁵N-enrichment and Ile-δ1, Leu, and Val ¹³CH₃ methyl labeling. The fusion protein was hydrolyzed by Kex2 protease at the recognition site Lys-Arg between residues ?29 and ?28 of the prosequence, producing the enzyme form called r27RCL. Here we report extensive backbone ¹H, ¹⁵N, and ¹³C, as well as Ile-δ1, Leu, and Val side chain methyl, NMR resonance assignments for r27RCL.     

Stopped-flow kinetic analysis of the interaction of anthraquinone anticancer drugs with calf thymus DNA, poly[d(G-C)].poly[d(G-C)], and poly[d(A-T)].poly[d(A-T)]

The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (1), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)]2, and poly[d(A-T)]2 have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)]2 and poly[d(A-T)]2 can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)]2 complexes are approximately 10 times lower than for the poly[d(A-T)]2 complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)]2 than from poly[d(A-T)]2, but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove.     

Histidine-15 and lytic activity of lysozyme

The literature data on the activity of histidine-15 modified hen egg white lysozyme are conflicting: the modified enzyme is reported to have more activity, similar activity or less activity by different authors. Amino acid analysis had shown modification of the single His-15. Detailed activity studies on His-15-modified (by iodoacetic acid or diethyl pyrocarbonate) lysozyme have shown that the contradicting reports are due to the specific choices of ionic strengths and cell wall substrate concentrations and can be attributed to the substrate being negatively charged. Our analysis suggests that even though histidine-15 is far removed from the active site of lysozyme, its chemical modification or binding of the negatively-charged substrate near it, changes the conformation around the active site. However, the change in the optimum activity on chemically modifying His-15 is small.     

Nucleotide sequence evidence of the unicentric origin of the βC mutation in Africa

Summary The origin of the ^C mutation was studied by characterizing nucleotide sequence polymorphisms on ^C chromosomes of patients from various African countries. In the majority of cases, the ^C mutation was found in linkage disequilibrium with a single chromosomal structure as defined by classical RFLP haplotypes, intergenic nucleotide sequence polymorphisms immediately upstream of the -globin gene, and intragenic -globin gene polymorphisms (frameworks). In addition, three atypical variant chromosomes carrying the ^C mutation were observed, and are most probably explained either by a meiotic recombination (two cases) or by one nucleotide substitution occurring in an unstable array of tandemly repeated sequences (one case). These data demonstrate the unicentric origin of the ^C mutation in central West Africa, with subsequent mutational modification in a small number of instances. The data also supports gene flow of the ^C chromosome from subsaharan Africa to North Africa.     

A novel beta thalassemia gene with a single base mutation in the conserved polypyrimidine sequence at the 3' end of IVS 2

An adult Algerian patient with homozygous beta thalassemia was found to have a unique beta thalassemia gene. Cloning and sequencing revealed that the only abnormality present in this beta gene is a transversion in the polypyrimidine stretch at the 3' end of the large intervening sequence (IVS 2) six bases 5' to the consensus AG dinucleotide sequence (CCGCCCACAG instead of CCTCCCACAG). In addition, digestion of the cloned fragment by the enzyme Mnl I demonstrates the disappearance of a restriction site as expected. This is the first example of a defect in the consensus sequence at the 3' end of an IVS leading to a thalassemia phenotype presumably due to decreased splicing.     

A simple strategy to amplify specifically the HLA-DQ beta gene region with genomic DNA as template

The nature of codon 57 in the HLA-DQ beta gene was recently reported as a potential marker of genetic susceptibility to insulin-dependent diabetes mellitus. When exploring the relevance of this marker by using genomic DNA amplification, we encountered difficulties resulting from the coamplification of the homologous DX beta region. A simple strategy is proposed to amplify the DQ beta region exclusively. It involves the preliminary digestion of genomic DNA with a restriction enzyme which cleaves DX beta specifically, leaving intact the DQ beta sequence. The amplified material is suitable for dot blot analysis and restriction enzyme digestion. This strategy is of general interest when homologous sequences impair the specificity of enzymatic DNA amplification.     

Stopped-flow kinetic, 1H, and 31P NMR analysis of the intercalation of ethidium with poly d(G-C) X d(G-C)

In a low salt buffer (0.011 M Na+) stopped-flow kinetic results for the SDS driven dissociation of an ethidium-Poly d(G-C) X d(G-C) complex are 8.7, 23, and 58.5 s-1 at 20, 30, and 40 degrees C, respectively. These results predict that in NMR experiments at high field strengths, ethidium should be in slow exchange among polymer binding sites. This has been found to be the case for both 31P (109 MHz) and 1H (imino proton spectra in H2O at 270 MHz) experiments. At higher salt, and/or higher temperature, and/or lower field, the bound and free peaks are no longer resolved in the NMR spectra. Good agreement is obtained between the stopped-flow kinetic results and the coalescence temperature observed in NMR experiments. Imino protons in base pairs on both sides of the intercalated ethidium are shifted approximately one ppm upfield while only the phosphate groups at the intercalation site experience large chemical shifts.