Results of collaborative study regarding the standardization of the Y-linked STR system DYS385 by the European DNA Profiling (EDNAP) group.

Y-chromosome linked short tandem repeat (STR) loci are inherited as a closely linked haplotype, which appears to remain stable in a given paternal lineage over many generations. In forensic cases, Y-linked STRs are particularly useful for the identification of human remains as well as in rape cases with mixed male/female stain samples. DYS385 is derived from tandemly duplicated segments of the Y chromosome thus giving rise to two fragments of variable length which do not behave like alleles but genotypes. The European DNA Profiling (EDNAP) group has carried out a collaborative exercise among 14 participating laboratories using DYS385 for typing of five unknown bloodstains and a control sample. Furthermore, population data from eight different European countries with samples sizes between 91 and 150 male individuals were collected. The results confirm previous observations that DYS385 is one of the most informative Y-linked STR loci. It could also be demonstrated that reproducible results can be obtained independently from the electrophoretic separation and detection methods used. Thus DYS385 may serve as a useful complementation to the routinely used autosomal STR systems in special cases.

Report of the European DNA Profiling Group (EDNAP)--an investigation of the hypervariable STR loci ACTBP2, APOAI1 and D11S554 and the compound loci D12S391 and D1S1656.

This paper describes the results of three collaborative exercises which continues the EDNAP theme to explore whether uniformity of DNA profiling results could be achieved between European laboratories using STRs. In an earlier exercise, complex hypervariable AAAG-repeat STR loci were investigated, but reproducibility was found to be poor because of the variation of techniques used by participating laboratories. In the exercise reported here, an internal allelic ladder composed of ACTBP2 and D11S554 fragments was distributed. This ladder was used to size ACTBP2 analysed by a "singleplex" PCR amplification and D11S554 combined with APOAI1 in a separate "duplex" reaction. Laboratories were asked to test 7 blood stains, one of which was a known control, and to report the results to the co-ordinating laboratory. The exercise demonstrated that ACTBP2 showed good reproducibility between laboratories, whereas further testing would be needed to validate APOAI1 and D11S554 for interlaboratory comparisons. In separate exercises, the simple loci D12S391 and D1S1656 were tested; both of these showed excellent reproducibility between laboratories.

Reproducibility of mtDNA analysis between laboratories: a report of the European DNA Profiling Group (EDNAP).

The aim of this collaborative exercise was to determine whether uniformity of mtDNA sequencing results could be achieved among different EDNAP laboratories. Laboratories were asked to sequence mtDNAHV1 region (16024-16365) from three bloodstains, proceeding in accordance with the protocol and strategies currently used in each individual laboratory. Cycle sequencing was used by 11 laboratories and solid phase single stranded sequencing was used by one laboratory. Different PCR strategies and PCR conditions were used by the different laboratories. Three laboratories used semi-nested PCR, two nested PCR, three direct amplification of HV1 and four amplification of overlapping fragments covering the HV1 region. Despite the diversity of methodologies used, all the laboratories reported the same results. The successful result of this exercise shows that PCR based mtDNA typing by automated sequencing is a valid, robust and reliable means of forensic identification despite the different strategies and methodologies used by the different laboratories.

A pentaplex automated fluorescent typing system for forensic identification and French Caucasian population data.

The polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci has already proven to be a method of choice for large scale typing of DNA samples in which the conventional restriction fragment length polymorphism (RFLP) technique is ineffective. A quadruplex PCR including HUMvWFA31A, HUMF13A01, HUMTH01, and HUMFESFPS STR loci is used successfully for routine forensic applications in our laboratory. However, the need to increase the discrimination power of the PCR systems used prompted us to develop a second system of a pentaplex PCR for the analysis of 4 additional STR loci (HUMD8S1179, HUMD18S51, HUMD21S11, and HUMFIBRA) and the sex determination by amplification of a segment of the X-Y homologous Amelogenin gene. Allele and phenotype frequencies for these 4 STR systems were obtained by multiplex amplification, from approximately 200 randomly selected and unrelated French Caucasian individuals. Statistical calculations for these phenotype distributions met expectations for Hardy-Weinberg equilibrium. Furthermore, the French allelic frequencies of D18S51, D21S11, and HUMFIBRA loci were compared with the data obtained by the Forensic Science Service (UK) for the British Caucasian population and proved to be similar.

Report of the European DNA profiling group (EDNAP): an investigation of the complex STR loci D21S11 and HUMFIBRA (FGA).

This paper describes a collaborative exercise which was intended to demonstrate whether uniformity of DNA profiling results could be achieved between European laboratories using two complex short tandem repeat (STR) loci. The loci D21S11 and HUMFIBRA (FGA) were chosen because they are commonly used by different European laboratories. D21S11 has approximately 14 common alleles (f > 0.001), whereas HUMFIBRA has 19 common alleles. Laboratories were asked to test seven blood stains, one of which was a known control, and to report the results to the coordinating laboratory. The exercise demonstrated that complex STRs were amenable to standardisation.

Report on the third EDNAP collaborative STR exercise. European DNA Profiling Group.

This report describes an inter-laboratory exercise completed on behalf of the European DNA Profiling (EDNAP) group. The exercise is one in a series designated to identify STR loci which could be used for harmonisation between participating European forensic science laboratories. Participants were asked to identify the alleles present in five bloodstains at the STR loci HUMTHO1 and HUMVWFA31/A. Two of the stains were prepared from mixtures of two different blood samples. There were no special instructions and each laboratory was requested to use the methodology normally employed for crime case investigations. All participating laboratories achieved the same results for both loci. In addition, the laboratories were also requested to report the results obtained from any other loci which would normally be used in crime case investigations. A comparison of these results showed some inter-laboratory variation.

First isolation of tandemly repeated DNA sequences in New World vultures and phylogenetic implications.

A highly repeated DNA sequence composed of closely related subunits that ranged from 171 to 176 base pairs has been cloned and characterized in the king vulture (Sarcoramphus papa). Related sequences were also isolated in the black vulture (Coragyps atratus). This new family of avian repetitive DNA elements is here termed the "HaeIII family." Genomic DNAs from a number of avian species were probed with one of the king vulture restriction fragments. In the cathartids, the hybridization patterns showed no individual or sexual variations. A strong HaeIII ladder was present in the two aforementioned species as well as in the Andean condor (Vultur gryphus), but in the black vulture the bands of the ladder alternated in intensity. Weaker hybridization signals were obtained in two ciconids, the jabiru stork (Jabiru mycteria) and the white stork (Ciconia ciconia). The HaeIII repeat was not detected in accipitrid birds of prey, a Polyborinae falconid, pelecanids, and psittacids.

French Caucasian population data obtained from fluorescently detected HUMvWFA31/A and HUMF13A01 short tandem repeat loci.

Allele and phenotype frequencies for two tetranucleotide STR (short tandem repeat or microsatellite) systems, HUMvWFA31/A and HUMF13A01, were obtained from a sample of approximately 240 unrelated individuals randomly selected from the French Caucasian population. PCR (polymerase chain reaction) products were analysed on 6% polyacrylamide denaturing gels and visualized using fluorescently labelled primers on the automated 373A ABI DNA sequencer (Applied Biosystems Inc.). French Caucasian allele frequencies were compared to other published Caucasian data. Conditions were optimised for the quadruplex PCR amplification of these two STR loci together with the HUMFESFPS and HUMTH01 loci and the quadruplex PCR was also performed on various forensic DNA samples.

French Caucasian population data for HUMTH01 and HUMFES/FPS short tandem repeat (STR) systems.

The recent technology of amplification of DNA sequences by the polymerase chain reaction (PCR) has already proved to be a very useful tool for the analysis of variable number of tandem repeat (VNTR) loci. Short tandem repeat (STR) loci appear as other promising PCR-based identification systems. In fact, DNA typing based on PCR amplification of STRs is very sensitive and allows to overcome major problems encountered when using the RFLP method, such as typing of very small amounts of DNA, highly degraded DNA or mixtures of DNA from more than one individual. Two STR systems, HUMTH01 (a tetranucleotide repeat (AATG) sequence located on chromosome 11) and HUMFES/FPS (a tetranucleotide repeat (ATTT) sequence located on chromosome 15) were investigated in order to determine allele and genotype frequencies for a French caucasian population sample. HUMTH01 and HUMFES/FPS alleles were amplified by the use of PCR and amplified STR sequences were analyzed on 6% Hydrolink Long Ranger gels and visualized by silver staining. The study was conducted on a sample of unrelated individuals (N approximately 190) randomly selected from the French caucasian population. The genotype distributions met Hardy-Weinberg expectations for both HUMTH01 and HUMFES/FPS STR systems. Furthermore, an additional allele, never reported before was observed at the HUMFES/FPS locus: it migrates as an allele containing 7 repeat units and corresponds to the smallest allele identified for this locus.

Medico-legal investigations of the Airbus, A320 crash upon Mount Ste-Odile, France.

The authors present the medico-legal investigations and identification after the aircrash of the Airbus A320 upon the Mount Sainte-Odile (France). The identification team comprising investigators from the gendarmerie, forensic pathologists, odontologists, and scientists of the Institute from Legal Medecine rapidly retrieved and identified 85 of the 87 victims, with 17 being identified through DNA typing, three through fingerprints and the remaining through dental records and specific physical or X-ray findings. Full autopsies were performed on all fatalities to determine patterns of injury and cause of death. Results lead us to point out the importance of a multidisciplinary team of forensic practitioners especially trained for managing medico-legal investigation in mass disaster and the ability of DNA technology to solve complex identification problems.

DNA fingerprinting from tissues after variable postmortem periods.

DNA typing is a useful tool in forensic cases for determining the identity of remains of humans who have been dead for various periods of time. DNA fingerprinting can be achieved only if high molecular weight DNA (HMWDNA) is extracted from the tissue samples of the bodies even after a long postmortem delay. Analyses were performed on various tissues collected during forensic autopsies of 24 bodies known postmortem ages. Tissues such as blood and kidney were found to be unsuitable for DNA fingerprinting because of a rapid degradation of the DNA after a period of one week. HMWDNA could be successfully extracted from brain cortex regardless of postmortem age.

Sex determination of forensic samples: co-amplification and simultaneous detection of a Y-specific and an X-specific DNA sequence.

The detection of restriction fragment length polymorphisms (RFLP) (1) in DNA extracted from forensic samples remains impossible in a significant number of cases due to deterioration and contamination of the biological material and the extremely low quantities of DNA isolated. The polymerase chain reaction (PCR) is a recent and particularly convenient method for analysing and typing very small amounts (10-20 ng) of degraded human DNA. DNA analysis at the level of a few cells present in forensic samples such as bloodstains, semen stains, vaginal swabs and head hair bulbs now appears possible using DNA amplification. A PCR protocol was adapted to simultaneously amplify a Y-specific DNA repeat sequence from the DYZ1 locus and an X-specific DNA repeat sequence from the DXS424 locus. The co-amplified Y-specific DNA fragment (102 bp) and X-specific DNA fragments (181-199 bp) were visualized on an ethidium bromide-stained 4% agarose gel. The male or female type of the amplified DNA extracted from blood samples, bloodstains, semen stains, vaginal swabs, brain tissue and 1, 2, 5, or 10 head hair bulbs was determined.

Variations during leaf development of the relative amounts of two bean (Phaseolus vulgaris) chloroplast tRNAs(Phe) which differ in their minor nucleotide content.

Bean (Phaseolus vulgaris cv. Saxa) chloroplasts contain two tRNA(Phe) species, namely tRNA(Phe)1 and tRNA(Phe)2. By sequence determination, we show that tRNA(Phe)2 is identical to the previously sequenced tRNA(Phe)1 except for two undermodified nucleotides. By reversed-phase chromatography analyses, we demonstrate that the relative amounts of these two chloroplast tRNAs(Phe) vary during leaf development: in etiolated leaves the undermodified tRNA(Phe)2 only represents 15% of total chloroplast tRNA(Phe), during development and greening it increases to reach 60% in 8-day-old leaves, and it then decreases to 9% in senescing leaves.

Codon recognition mechanisms in plant chloroplasts.

In chloroplasts, all 61 sense codons are found in chloroplast (cp) DNA sequences coding for proteins. However among the sequenced cp tRNAs or tRNA genes, tRNAs with anticodons complementary to codons CUU/C (Leu), CCU/C (Pro), GCU/C (Ala) and CGC/A/G (Arg) [or CGC/A (Arg) in Marchantia] have not been found. In this paper we show that cp tRNA(Ala)(U*GC) cp tRNA(Pro)(U*GG) and cp tRNA(Arg)(ICG) are able to decode the corresponding four-codon family. In the case of leucine codons CUU/C, we show that 'U:U and U:C wobble' mechanisms can operate to allow the reading of these codons by cp tRNA(Leu)(UAm7G).

Preparation of a tRNA-dependent wheat germ protein-synthesizing system.

A method is described for the preparation of a tRNA-dependent wheat germ protein-synthesizing system. This system can be supplied with exogenous tRNAs of eukaryotic or prokaryotic origin. In order to obtain maximal aminoacylation of the added tRNAs, the translation assays can be supplemented with homologous aminoacyl-tRNA synthetase preparations. Such a tRNA-dependent wheat germ protein-synthesizing system, which is easy to prepare, can be used not only to translate plant cytoplasmic mRNAs in the presence of added cytoplasmic tRNAs, but also to determine the translation activity of exogenous tRNAs from various sources in the presence of either natural or in vitro synthesized mRNAs.

Adjustment of the tRNA population to the codon usage in chloroplasts.

In chloroplasts there is a correlation between the amounts of tRNAs specific for a given amino acid and the codons specifying this amino acid. Furthermore, for the amino acids coded for by more than one codon, the population of isoaccepting tRNAs is adjusted to the frequency of synonymous codons used in chloroplast protein genes. A comparison by two-dimensional gel electrophoresis of the tRNA populations extracted from chloroplasts and from chloroplast polysomes shows that all chloroplast tRNAs are involved in protein biosynthesis.

On the incidence of malignant tumors in routine autopsies.

The present study taken over a 12-year time period analyzes malignant tumors in a large number of autopsies, with specific consideration of the frequency and location, age and sex group distribution, and histologic classification of the tumors. The tumors were first evaluated in terms of specific organs and organ systems and were then histologically classified. On a total of 23338 subjects who died between the years 1960 and 1971, 17052 autopsies were carried out (73.1%) and only 15384 (65.9%) of these were evaluated for out study. Altogether we located 4911 cases of MT, i.e., 31.9% of the evaluated autopsies. The number of MT in the males slightly exceeded that in the females (2508[51.1%] MT and 2403 [48.9%] MT, respectively.) We calculated over 60% of the MT to be in the 7th and 8th age groups. By comparing the two time periods (1960-1965) and 1966-1971) we calculated a significant increase in the MT rate by 1.7%. This increase was found mainly to effect the males (the female group showed only a slight increase). The greatest number of MT were located in the digestive system with an MT rate of 26.2%; next came the urogenital system (23.4%) and then the respiratory system (18.7%). The respiratory system took first position of importance in the males with an MT rate of 30.3% followed by the digestive system with 27.4%. The urogenital system dominated in the females with a rate of 29.0%. The rate of MT of the respiratory system was significantly higher in the males...