The HNF1A mutant Ala180Val: Clinical challenges in determining causality of a rare HNF1A variant in familial diabetes.

AIMS: Heterozygous mutations in hepatocyte nuclear factor-1A (HNF1A) cause maturity-onset diabetes of the young type 3 (MODY3). Our aim was to compare two families with suspected dominantly inherited diabetes and a new HNF1A variant of unknown clinical significance. METHODS: The HNF1A gene was sequenced in two independently recruited families from the Norwegian MODY Registry. Both familes were phenotyped clinically and biochemically. Microsatellite markers around and within the HNF1A locus were used for haplotyping. Chromosomal linkage analysis was performed in one family, and whole-exome sequencing was undertaken in two affected family members from each family. Transactivation activity, DNA binding and nuclear localization of wild type and mutant HNF-1A were assessed. RESULTS: The novel HNF1A variant c.539C>T (p.Ala180Val) was found in both families. The variant fully co-segregated with diabetes in one family. In the other family, two subjects with diabetes mellitus and one with normal glucose levels were homozygous variant carriers. Chromosomal linkage of diabetes to the HNF1A locus or to other genomic regions could not be established. The protein functional studies did not reveal significant differences between wild type and variant HNF-1A. In each family, whole-exome sequencing failed to identify any other variant that could explain the disease. CONCLUSIONS: The HNF1A variant p.Ala180Val does not seem to cause MODY3, although it may confer risk for type 2 diabetes mellitus. Our data demonstrate challenges in causality evaluation of rare variants detected in known diabetes genes.

Characterization of HIV-1 reverse transcriptase with antibodies indicates conformational differences between the RNAse H domains of p 66 and p 15.

Antibody binding to the p 66 and p 15 RNase H regions of HIV-1 reverse transcriptase was compared using a polyclonal rabbit immune serum raised against a synthetic peptide from the RNase H region of reverse transcriptase (aa 511-527) and six monoclonal antibodies binding to discontinuous epitopes in the RNase H region of p 66. The antigens used in Western blot analysis included recombinantly expressed homodimeric p 66 digested with the HIV-1 protease for generation of the p 51 and p 15 polypeptides and two different length RNase H domains expressed as Trp E fusion proteins (aa 410-560 and aa 441-560). The polyclonal rabbit antibody binding to a continuous epitope recognized both the Trp E-fusion proteins and also the polypeptides p 66 and p 15 generated by processing of homodimeric p 66 with the viral protease. Two additional cleavage products with estimated molecular weights of 9 and 11 kDa were also detected. The anti-RNase H MAbs binding to discontinuous epitopes recognized only the RNase H domain of the p 66 polypeptide and the Trp E-RNase H fusion protein when this was expressed together with the C-terminal part of the polymerase domain. The results indicate conformational differences between the RNase H domain of the p 66 subunit and the RNase H p 15 polypeptide.

Application of magnetic beads in bioassays.

This review outlines the possibilities of magnetic separation techniques and the application of magnetic beads in bioassays. By linking monoclonal antibodies or DNA to magnetic beads, or by using magnetic beads coated with streptavidin, a specific interaction with the corresponding target is ensured. By means of an external magnet, the recovery of material for further studies is greatly simplified. Magnetic beads have proven valuable in cell separations, for example, removal of tumor cells from bone marrow and isolation of lymphoid cells from peripheral blood, and for the isolation, identification and genetic analysis of specific nucleic acid sequences (DNA or RNA) and for isolation of DNA binding proteins. In addition, some of these techniques have also proven to be useful in the detection of specific nucleic acids from viruses or bacteria.

Purification to homogeneity and characterization of a redoxyendonuclease from calf thymus.

A redoxyendonuclease from calf thymus was purified to apparent homogeneity. The redoxyendonuclease recognized and induced cleavage of DNA damaged by ultraviolet light. The enzyme preparation produced a single band of a relative molecular mass of approximately 34 kDa upon SDS/PAGE. The apurinic/apyrimidinic endonuclease and the DNA glycosylase activities remained associated in the apparently homogeneous preparation of the enzyme. The redoxyendonuclease activity displayed a broad pH optimum between pH 5.0-8.5 and exhibited no requirement for divalent cations. By application of FPLC columns Mono-S, Mono-Q and Mono-P, the isoelectric point (pI) of the enzyme was found to be approximately 8.0. Using the DNA sequencing procedure of Maxam and Gilbert [Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560] the purified enzyme was found to incise ultraviolet-light-irradiated DNA at pyrimidine sites as observed previously with a more crude form of the enzyme. While the most frequently cleavaged sites for the crude preparation were at cytosine residues, the apparently homogeneous enzyme preparation frequently induced cleavage sites at both cytosine and guanine residues. Predominant incision induced by the apparently homogeneous preparation was observed at guanine residues when a particular DNA sequence was used as substrate. Furthermore, the 16 N-terminal amino acid residues of the purified enzyme were identified. The sequence did not show any significant similarity to other known proteins.

Action of spermidine, N1-acetylspermidine, and N8-acetylspermidine at apurinic sites in DNA.

The cleavage efficiency of spermidine and its acetyl derivatives (N1-acetylspermidine and N8-acetylspermidine) at apurinic sites in DNA were examined by PAGE-urea analysis. The three polyamines induced different rates of cleavage when compared at 1 mM concentrations. The order of effectiveness were: spermidine greater than N8-acetylspermidine greater than N1-acetylspermidine. Thus a decrease in efficiency was observed when the first order amino-groups of spermidine were blocked. The N-8amino-group of spermidine was less effective in inducing cleavage at AP-sites than the N1-amino-group. Among several proposed models of polyamine-DNA interactions, our results can best be explained by the model postulated by Liquori et al.

Applications of magnetic beads with covalently attached oligonucleotides in hybridization: isolation and detection of specific measles virus mRNA from a crude cell lysate.

A novel, rapid, one-step isolation procedure utilizing oligonucleotides end-attached to magnetic beads (Dynabeads) has been developed. The beads and their captured target nucleic acids were separated after 2 h from the hybridization solution using an external magnet. This procedure was used to isolate measles virus hemagglutinin (H) mRNA from infected cells dissolved in 5 M guanidine thiocyanate. The yield of the specific, undegraded H-mRNA was found to be near the estimated total amount of H-mRNA present in the cells. The magnetic beads were also used for detection of measles virus H-mRNA in a crude cell lysate by sandwich hybridization. The experimental detection limit was 630 amol H-mRNA.

Mechanism of incision by an apurinic/apyrimidinic endonuclease present in human placenta.

An apparently homogeneous enzyme preparation, containing an apurinic/apyrimidinic (AP) endonuclease from human placenta, was by DNA sequencing analysis found to act as a class I AP-endonuclease, i.e. produce a 3'-deoxyribose and 5'-phosphomonoester nucleotide termini.

Mechanism of spontaneous loss of heat-stable toxin (STa) production in enterotoxigenic Escherichia coli.

Five strains of enterotoxigenic Escherichia coli (ETEC) showing spontaneous loss of heat-stable enterotoxin (STa) production were studied to elucidate the underlying genetic mechanisms. Southern blot analysis revealed that loss of STa production, and the corresponding lack of hybridization with the STa gene probes, were associated with deletions of DNA fragments harboring the relevant toxin genes rather than with loss of plasmids.

Action of a mammalian AP-endonuclease on DNAs of defined sequences.

An apurinic/apyrimidinic (AP) specific endonuclease from mouse plasmacytoma cells (line MPC-11), was observed to cleave apurinic sites in oligonucleotides 9, 11, 12, 39 and 40 nucleotides in length. However, the enzyme failed to cleave AP-sites in two oligonucleotides 7 nucleotides in length. The maximum rates of digestion observed on these short single-stranded DNA (ssDNA) fragments were approximately 1/30 of the rates observed on double-stranded DNA (dsDNA). In studies using the Maxam-Gilbert DNA sequencing analysis, apurinic sites in purine-rich regions were preferentially cleaved in dsDNA but not in ssDNA, indicating that the enzyme has a sequence preference on dsDNA. These results suggest that some sites on DNA might be more efficiently repaired than others.

Analysis of cleavage products of DNA repair enzymes and other nucleases. Characterization of an apurinic/apyrimidinic specific endonuclease from mouse plasmacytoma cells.

We have developed a strategy by which the nature of phosphodiester bond breaks produced by various DNA-repair endonucleases and also other nucleases, can be characterized. A purified apurinic/apyrimidinic (AP) specific endonuclease from a permanently established mouse plasmacytoma cell-line (MPC-11) has been examined with respect to the exact incision site generated at the baseless site. By the aid of enzymatic treatment with calf intestinal phosphatase, the 3'-phosphatase activity of T4-polynucleotide kinase, chemical modification with piperidine in addition to the Maxam-Gilbert sequencing procedure, followed by separation on a DNA-sequencing gel, the nature of the cleaved phosphodiester bond, both 3' and 5' to the cleavage site, has been established. The AP-specific endonuclease investigated was classified as a class II AP-endonuclease according to the four possible classes of AP-endonuclease with respect to the termini produced. By use of this technique each single damaged and cleaved site can be investigated separately.

Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes.

We present a study on the refinement of filter-hybridization conditions for a series of synthetic oligonucleotides in the range from 17 to 50 base residues in length. Experimental conditions for hybridization and the subsequent washing steps of the filter were optimized for different lengths of the synthetic oligonucleotides by varying the formamide concentration and washing conditions (temperature and monovalent cation concentration). Target DNA was immobilized to the nitrocellulose filter with the slot blot technique. The sequences of the synthetic oligonucleotides are derived from the third exon of the human oncogene c-myc and the corresponding viral gene v-myc and the G + C content was between 43 and 47%. Optimal conditions for hybridization with a 82% homologous 30-mer and 100% homologous 17-, 20-, 25-, 30-, and 50-mers were found to be a concentration of formamide of 15, 15, 30, 30, 40, and 50%, respectively. Optimal conditions for washing were 0.5X standard sodium citrate (SSC) at 42 degrees C for 2 X 15 min. The melting temperature for these optimal hybridization and washing conditions was calculated to be up to 11 degrees C below the hybridization temperature actually used. This confirms that the duplexes are more stable than expected. The melting points for 17-, 20-, and 30-mers were measured in the presence of 5X SSC and found to be 43, 58, and 60 degrees C, respectively. Competition between double- and single-stranded DNA probes to the target DNA was investigated. The single-stranded DNA probes were about 30- to 40-fold more sensitive than the double-stranded DNA probes.

Comparative study of colony hybridization with synthetic oligonucleotide probes and enzyme-linked immunosorbent assay for identification of enterotoxigenic Escherichia coli.

On the basis of the published nucleotide sequences of the genes that code for the heat-labile toxin LTh and the heat-stable toxins STaI and STaII of human enterotoxigenic Escherichia coli, a 34-mer and two 33-mer oligonucleotide probes were synthesized. To compare their relative efficacies in the detection and differentiation of enterotoxigenic E. coli, a colony hybridization technique using these probes and a GM1 ganglioside enzyme-linked immunosorbent assay using monoclonal anti-LT and anti-ST antibodies were used with 76 strains of E. coli with known enterotoxin profiles. For further evaluation of probe specificity, the enterotoxigenic bacteria Vibrio cholerae O1 and non-O1 and Yersinia enterocolitica were examined with the colony hybridization technique. The sensitivity of colony hybridization compared favorably with that of GM1 ganglioside enzyme-linked immunosorbent assay, and the two assays showed a high level of concordance in specific detection and differentiation of E. coli with various enterotoxin profiles (kappa = 0.906, P less than 0.00001). The probes did not hybridize with DNAs from strains of V. cholerae O1 or non-O1 or Y. enterocolitica.

Substrate specificity of 3-methyladenine-DNA glycosylase from calf thymus.

3-Methyladenine-DNA glycosylase from calf thymus recognizes both 3-methyladenine (3-mAde), 7-methylguanine (7-mGua) and 3-methylguanine (3-mGua) residues in calf thymus DNA; the rate of release of 3-mAde is approximately eightfold higher than that for 7-mGua. The best DNA polymer substrates appeared to be those having an A-type helical conformation such as d(A-T)n and d(G-C)n. The Km values for release of 3-mAde and 7-mGua were approximately the same for the above mentioned two substrates whereas the Vmax for excision of 3-mAde was threefold higher than that of 7-mGua. The rate of hydrolysis of 7-mGua residues in d(G-C)n was similar to that found for the excision of 3-mAde in calf thymus DNA. The polymer d(G)n X d(C)m, which possesses a B-type helical conformation, was a poor substrate and the rate of excision here was approximately the same as with calf thymus DNA having the B-type structure. Polyamines greatly influenced the activity and at low concentrations a 50-100% increase in the release of 7-mGua, but not 3-mAde, was observed. With higher concentrations the rate of excision of both bases decreased sharply. The sequence specificity of the DNA glycosylase on naturally occurring DNA was studied using methylated DNA fragments from the plasmid pUC18. The results revealed that some 3-mAde as well as 7-mGua residues were seldom attacked. These 3-mAde residues were positioned either 5' to another Ade residue or in a stretch of pyrimidines, and the 7-mGua residue 3' to another Gua residue. The 3-mAde residue most frequently recognized was situated 3' to another Ade residue, and in the case of 7-mGua it was the central Gua residue in the sequence -G-G-G-.

Properties and mechanism of action of eukaryotic 3-methyladenine-DNA glycosylases.

3-Methyladenine-DNA glycosylase activities have been identified in all eukaryotic cell systems studied. Some of the results from these studies are reviewed here. The enzymes possess molecular weights between 24 X 10(3) and 34 X 10(3), they have a broad pH optimum at approximately pH 8, require double-stranded DNA and act in the absence of any cofactors. The enzyme can excise several different methylated bases from DNA such as 3-methyladenine, 7-methylguanine and 3-methylguanine. The specific activity of this DNA glycosylase in mouse L-cells was found to be a function of the proliferative state of the cell. In vitro quantification of this DNA repair activity in synchronized mouse L-cells suggests that it is regulated within a defined temporal sequence prior to the onset of DNA replication. Using DNA fragments of defined sequences it was observed that the efficiency of removal of the methylated bases is sequence-dependent.