Population variation of human mitochondrial DNA hypervariable regions I and II in 105 Croatian individuals demonstrated by immobilized sequence-specific oligonucleotide probe analysis.

AIM: To detect sequence variation in 105 Croatian individuals by the use of duplex polymerase chain reaction amplification of full-length hypervariable region I and II (HVI/HVII) products and subsequent hybridization to a linear array of 27 immobilized sequence-specific oligonucleotide (SSO) probes, which targets six regions within HVI and HVII, and two additional sites, 189 and 16093. METHODS: Chelex-extracted bloodstains were used for amplification of HV regions. In all cases, a single robust amplification was sufficient for immobilized SSO probe typing and subsequent direct sequence analysis for both HVI and HVII. This method, suitable for a range of forensic samples (including shaft portions of single hairs), was also applied to the analysis of 18 skeletal elements recovered from a mass grave. Using a panel of immobilized SSO probes, we have developed a rapid screening approach to mitochondrial DNA (mtDNA) haplotyping before direct sequence analysis. RESULTS: We established a reference sequence database of mtDNA haplotypes for 105 randomly selected Croatian individuals. Fifty different mitotypes were observed (33 unique). The most frequent mitotypes occurred 18 times or approximately 17.1% [111111 189 (A) 16093 (T)] and 11 times or approximately 10.5% [131111 189 (A) 16093 (T)]; all other mitotypes occurred 5% or less. The corresponding genetic diversity value for this database was approximately 0.952. The usefulness of establishing an mtDNA reference database with immobilized SSO probe testing has been demonstrated by determining the strength of a match comparison obtained for one skeletal element and a corresponding maternal reference from 18 specimens recovered from a mass grave. CONCLUSION: The sequence variation detected by the panel of immobilized SSO probes is sufficiently diverse to be used for identification of human skeletal remains from mass graves. The immobilized SSO typing strip targets polymorphic regions within HVI and HVII and is a useful identification tool for mass grave and mass disaster analysis, as well as for criminal casework testing.

The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age.

An immobilized sequence-specific oligonucleotide (SSO) probe system consisting of 16 SSO probes that detect sequence polymorphisms within five regions of the mtDNA control region was used to investigate the frequency of heteroplasmy in human mtDNA. Five regions of hypervariable region II (HVII) of the control region were studied in blood-, muscle-, heart-, and brain-tissue samples collected from 43 individuals during autopsy. An initial search for heteroplasmy was conducted by use of the SSO probe system. Samples in which multiple probe signals were detected within a region were sequenced for the HVII region, to verify the typing-strip results. The frequency of heteroplasmy was 5 of 43 individuals, or 11.6%. The frequency of heteroplasmy differed across tissue types, being higher in muscle tissue. The difference in the frequency of heteroplasmy across different age groups was statistically significant, which suggests that heteroplasmy increases with age. As a test for contamination and to confirm heteroplasmy, the samples were sequenced for the HVI region and were typed by use of a panel of five polymorphic nuclear markers. Portions of the tissues that appeared to be heteroplasmic were extracted at least one additional time; all gave identical results. The results from these tests indicate that the multiple sequences present in individual samples result from heteroplasmy and not from contamination.

Neurofibromatosis: review and report of case.

Neurofibromatosis, or von Recklinghausen's disease, is the result of a genetic mutation affecting more than 80,000 persons in the United States. Also known as the "Elephant Man" disease, it is marked by multiple pedunculated soft tumors distributed over the entire body associated with areas of pigmentation. This article discusses various aspects of the disease, and presents a report of classical neurofibromatosis.

The tRNAGlu2 gene in the rrnB operon of E. coli is a prerequisite for correct RNase III processing in vitro.

RNase III cleaves precursor 16S RNA and precursor 23S RNA from the ribosomal RNA transcript. In vitro transcription experiments, using plasmids with the rrnB operon truncated in the 16S RNA and with various deletions in the spacer tRNA region, showed that no matter what size of deletion if the tRNA gene was affected RNase III processing of 16S RNA became incomplete. In comparison to a control plasmid, where only the 16S RNA gene was truncated and that showed normal RNA processing, plasmids where the tRNA gene was deleted partially or totally either the 5' or the 3' end of 16S RNA was processed. This relation between RNase III processing and the 3-dimensional structure of tRNA suggests an interaction between RNase III and a tRNA processing enzyme most probably RNase P.

Microvascular injury distal to unilateral pulmonary artery occlusion.

We explored three questions: 1) does edema fluid accumulate distal to temporary unilateral pulmonary artery occlusion (TUPAO); 2) if so how rapidly does it accumulate; and 3) how is it affected by positive end-expiratory pressure (PEEP)? Using a tracheal divider we measured pulmonary capillary blood flow (Qc), tissue volume (Vt), and diffusing capacity (DLCO) in each lung with a rebreathing method. After control measurements in 12 dogs, the left pulmonary artery was occluded and measurements were repeated at intervals during 4 h of occlusion and 30 min after release of the occlusion. Six of the dogs were ventilated with 10 cmH2O PEEP. Finally the lungs were removed, weighed, and fixed for histology. TUPAO caused a 29% increase in Vt of the left lung without PEEP and a 59% increase with PEEP. After release of the occlusion, Qc and DLCO in the left lung returned to control levels within 30 min in dogs not on PEEP but remained depressed in dogs ventilated with PEEP even though PEEP was removed. At postmortem the left lung weighed more than expected in both groups of dogs but was significantly heavier in those on PEEP. Histology confirmed bronchovascular cuffing with edema and hemorrhage.