Thomas Kaufmann

The human complement component C8B gene : structure and phylogenetic relationship

The eighth component of human complement (C8) is a serum protein that consists of three chains (α, β and γ), encoded by three separate genes, viz., C8A, C8B, and C8G. In serum, the β-subunit is non-covalently bound to the disulfide-linked α-γ subunit. Using a full-length C8β cDNA probe, we isolated several clones from human genomic λ DNA libraries. Four λ clones covering the complete cDNA sequence were characterized by TaqI restriction mapping and were “shotgun” subcloned into M13. C8β-cDNA-positive clones were partially sequenced to characterize the 12 exons of the gene with sizes from 69 to 347 bp. All intron-exon junctions followed the GT-AG rule. By using polymerase chain reaction (PCR) primers located in the adjacent intron sequences, all 12 exons of the C8B gene could be amplified from genomic DNA. All fragments showed the expected sizes. The sizes of eight introns could be determined by using primer pairs that amplified two exons and the enclosed intron, and by restriction mapping. These analyses and the insert sizes of the genomic λ clones indicate that the C8B gene has a total size of approximately 40 kb. The polymorphic TaqI site of the C8B gene localized in intron 11 could be demonstrated by direct restriction fragment analysis of a PCR fragment containing exons 11 and 12, and the enclosed intron 11. Homology comparison of the C8B gene with C8A and C9 on the basis of the exon structure confirmed the ancestral relationship known from the protein level.

Quantitative and qualitative analysis of DNA extracted from postmortem muscle tissues

SummaryDNA extracted from 33 postmortem muscle specimens was analyzed using MZ 1.3, a hypervariable minisatellite probe, as well as locus-specific minisatellite probes (g3, MS1 and MS43). After storage at −25°C for 10 months, DNA from all the samples was partially (approximately 21% of total DNA) degraded even when autopsy was performed 1 day post mortem. However, more than 90% of DNA samples up to at least 3 days post mortem were suitable to obtain good restriction fragment length polymorphism (RFLP) patterns. When small strips of specimen were stored for 8 days at room temperature in moist chambers, approximately 42% of total DNA was degraded. Only 30% of these DNA samples still showed good RFLP patterns. However, no obvious relation between qualities of DNA analyzed by detection of RFLP and quantities of total and high-MW DNA became apparent. A case of familial relationship was ascertained by DNA fingerprints. Since DNA of good quality can be recovered from muscle tissues in large quantities, DNA extraction from muscle tissues and detection of RFLP patterns should be very useful for individual identification in autopsy cases.ZusammenfassungGenomische DNA wurde aus 33 Muskelgewebsproben postmortal extrahiert and im Southern Blot-Verfahren sowohl mit der Multi-locus-Minisatelliten-DNA-Sonde MZ 1.3 als auch mit Locus-spezifischen DNA-Sonden (g3, MS1 and MS43) analysiert. Nach Lagerung der Gewebsproben bei −25°C fur 10 Monate war die DNA von allen Proben partiell degradiert (ca. 25% Anteil degradierter DNA an Gesamt-DNA), auch wenn die Obduktion innerhalb eines Tages nach Eintritt des Totes erfolgte. Dennoch waren über 90% der DNA-Proben, die bis zu drei Tage nach Tod entnommen werden waren, für eine Southern Blot-Analyse geeignet. Bei Lagerversuchen von kleinen Muskelgewebsproben bei Raumtemperatur in einer feuchten Kammer fur acht Tage waren ca. 42% der Gesamt-DNA degradiert. Nur von 30% dieser gelagerten DNA-Proben waren noch Minisatelliten-DNA-Fragmente darzustellen. Es war jedoch keine deutliche Abhängigkeit zwischen dem jeweiligen Anteil von hochmolekularer DNA zu Gesamt-DNA einer Probe and der Detektion von Minisatelliten-DNA-Fragmenten zu erkennen. Zusätzlich konnte in einem Fall eine Verwandtschaftsbeziehung anhand der Minisatelliten-DNA-Fragmente mit MZ 1.3 untersucht werden. Da insgesamt genomische DNA in guter Qualität und ausreichender Menge aus Muskelgewebsproben isoliert werden konnte, erscheint diese Methode zur Darstellung von individuellen DNA-Fragmentmustern bei forensischen Fragestellungen in Autopsiefällen sehr nützlich.

Heterogeneity in the genetic basis of human complement C9 deficiency

Activation of the complement system leads to the formation of the membrane attack complex (MAC) which is composed of single molecules of the components C5b, C6, C7, and C8 as well as variable numbers of C9. The C9 multimer forms a membrane pore in target cells, thus leading to lysis and cell death (for review see Plumb and Sodetz 1998). Deficiency of one of the terminal complement components leads to incomplete formation of the MAC and therefore causes an increased susceptibility to recurrent Neisserial infections (Figueroa and Densen 1991). C9-deficient individuals are usually healthy (Figueroa and Densen 1991; Fukumori et al. 1989), as the initial steps in formingmthe MAC seem to be sufficient for cell death because C8 already penetrates the membrane (Harriman et al. 1981; Stolfi 1968). Cases of C9 deficiency with episodes of meningitis have been described (Nagata et al. 1989; Zoppi et al. 1990). C9 deficiency is apparently very rare in the Caucasian population, but more common in Orientals (Fukumori et al. 1989; Hayama et al. 1989). C9 is a single-chain glycoprotein of 538 amino acids and is encoded by a gene located on chromosome 5p13 (Abbott et al. 1989; Rogne et al. 1989). The gene is composed of 11 exons with lengths between 100 and 250 base pairs (bp) (Marazziti et al. 1988), except for exon 11 which extends over more than 1 kilobase (kb), as it includes the 3 untranslated region (UTR). In a previous study, we developed an approach to analyze each exon individually using exon-specific polymerase chain reaction (PCR) and direct sequencing. C9 deficiency in a Swiss family (Zoppi et al. 1990) was shown to be due to two independent point mutations creating stop codons. One is located in exon 2 at cDNA position 166, the other in exon 4 at cDNA position 464 (Witzel-SchloÈmp et al. 1997). As these mutations segregate independently, they are sufficient to explain the complete absence of serum C9 in two family members. In the present study, we investigated the genetic basis of inherited C9 deficiency in an adult of Irish origin reported previously as patient X (Hobart et al. 1997) and an unrelated Irish family. Family members studied were the index case Y, his brother, and two sisters, all of whom are over 50 years of age. The complement deficiencies of the index cases X and Y were identified because of the complete lack of alternative complement pathway activity. Further tests, as described for subject X (Hobart et al. 1997), showed them to be C9 deficient. One of Ys sisters is also C9-deficient. None of these probands reported any Neisserial infection, but a sister in family Y died at the age of 22 of meningitis, probably meningococcal. As proband X was shown to be heterozygous for C6, C7, and C9 DNA markers, he was expected to be a compound heterozygote (Hobart et al. 1997). To identify the genetic basis for the lack of C9 in these subjects, an individual primer pair was used to amplify each of the 11 exons (except for exon 1 and the 3 part of exon 11 which have not yet been analyzed as the exon-flanking intron sequences are still unknown). PCR was carried out using exon-specific primers as described (Witzel-SchloÈmp et al. 1997). Exons 2±11 and exon-flanking intron sequences are deposited in the EMBL data library (accession numbers Y08545 to Y08554). DNA from unrelated healthy individuals was used as control. Genomic DNA was obtained from whole blood samples according to standard procedures. The purified double-stranded PCR fragments were sequenced by dideoxy chain termination, using the Thermo Sequenase Cycle Sequencing Kit (Amersham Life Science, Cleveland, Ohio). K. Witzel-SchloÈmp ? C. Rittner ? T. Kaufmann ? P.M. Schneider ( ) Institute of Legal Medicine, Johannes Gutenberg University, Am Pulverturm 3, D-55131 Mainz, Germany

The eighth component of human complement: molecular basis of C8A (C81) polymorphism

Using an exon-specific polymerase chain reaction (PCR) followed by direct DNA sequence analysis we have analyzed the polymorphism of the α-chain of the eighth component of human complement (C8) at the DNA level. We found that two common alleles, C8A*A and C8A*B, are characterized by the substitution of a single amino acid (Gln to Lys), which is caused by a point mutation of a single nucleotide (C to A) in exon 3 at position 187 of the mature C8α cDNA sequence. Based on this mutation, an allele-specific PCR was designed detecting the two alleles of C8A. We applied this method to type the C8A polymorphism using DNA samples from a Chinese Han population. The comparison with the data of protein typing of the same samples proved that the described method is efficient and reliable for the identification of C8A genotypes and may be valuable for further application in population studies and forensic science.

Further development of a commercial driving simulation for research in occupational medicine

ObjectivesThe purpose of this study was to refine a commercial car driving simulation for occupational research. As the effects of ethanol on driving behavior are well established, we choose alcohol as a test compound to investigate the performance of subjects during simulation.Materials and MethodsWe programmed a night driving scenario consisting of monotonous highway and a rural road on a Foerst F10-P driving simulator. Twenty healthy men, 19–30 years, participated in a pilot study. Subjects were screened for simulator sickness, followed by training on the simulator one hour in total. Experiments were performed in the morning on a separate day. Participants were randomized into either an alcoholized or a control group. All subjects drove two courses consisting of highway and rural road and were sober for the first course. During a 1 h break the ethanol group drank an alcoholic beverage to yield 0.06% blood alcohol concentration (BAC). Generalized linear mixed models were used to analyze the influence of alcohol on driving performance. Among others, independent variables were Simulator Sickness Questionnaire scores and subjective sleepiness.ResultsSubjects did not experience simulator sickness during the experiments. Mean BAC before the second test drive was 0.065% in the mildly intoxicated group. There was no clear-cut difference in the number of crashes between 2 groups. BAC of 0.1% would deteriorate mean braking reaction time by 237 ms (SE = 112, p < 0.05). Ethanol slightly impaired the tracking in the right-hand curves (p = 0.058). Braking reaction time improved by 86 ms (SE = 36, p < 0.05) for the second test drive, indicating a learning effect.ConclusionsIn sum, a clear ethanol effect was observed in the driving simulation. This simulation seems suitable for occupational research and produces little simulator sickness. Controlling for possible learning effects is recommended in driving simulation studies.

Reliability of Restriction Enzyme Digestions of Genomic DNA for the Generation of DNA Fingerprints

Since minisatellite DNA probes are used for the detection of hypervariable loci in eucaryotic genomes [1] the application of so called DNA fingerprints and DNA technology itself in paternity testing and forensic casework is critically discussed ([3]; Brinkmann et al., this volume). A particular problem is the possibility of obtaining partially digested genomic DNA in casework after treatment with restriction enzymes leading to inconclusive or even false results. This is even more important when multilocus DNA probes are used, since the total number of fragments in a given person is not known in advance. But also with single locus probes, where only two allelic fragments are usually detected, a fragment shift of additional bands may be produced by partially digested DNA.

Rapid and standardized quantitation of hemolytic activity of the fourth component of human complement.

Based on a method that uses the fourth component of complement (C4)-deficient guinea pig serum to quantify the hemolytic activity of C4, we developed an assay that allows the processing of a large number of individual samples with high reproducibility. In contrast to the conventional procedure using titration curves of each sample to be determined, we can show that a single appropriate dilution of the sample allows accurate quantitation of hemolytic activity. The reliability of the procedure is demonstrated using either C4A- or C4B- deficient and normal individual samples.