Characterization of ancestral and derived Y-chromosome haplotypes of New World native populations

We analyze the allelic polymorphisms in seven Y-specific microsatellite loci and a Y-specific alphoid system with 27 variants (alphah I-XXVII), in a total of 89 Y chromosomes carrying the DYS199T allele and belonging to populations representing Amerindian and Na-Dene linguistic groups. Since there are no indications of recurrence for the DYS199C-->T transition, it is assumed that all DYS199T haplotypes derive from a single individual in whom the C-->T mutation occurred for the first time. We identified both the ancestral founder haplotype, 0A, of the DYS199T lineage and seven derived haplogroups diverging from the ancestral one by one to seven mutational steps. The 0A haplotype (5.7% of Native American chromosomes) had the following constitution: DYS199T, alphah II, DYS19/13, DYS389a/10, DYS389b/27, DYS390/24, DYS391/10, DYS392/14, and DYS393/13 (microsatellite alleles are indicated as number of repeats). We analyzed the Y-specific microsatellite mutation rate in 1,743 father-son transmissions, and we pooled our data with data in the literature, to obtain an average mutation rate of.0012. We estimated that the 0A haplotype has an average age of 22,770 years (minimum 13,500 years, maximum 58,700 years). Since the DYS199T allele is found with high frequency in Native American chromosomes, we propose that 0A is one of the most prevalent founder paternal lineages of New World aborigines.     

Identification of war victims from mass graves in Croatia, Bosnia, and Herzegovina by use of standard forensic methods and DNA typing

The postmortem remains of sixty-one war victims were excavated from 6 mass graves in Bosnia and Herzegovina one and a half years after interment Using standard identification methods, including the matching of medical and dental records, the recognition of distinguishing characteristics such as the use of clothing and belongings, and video superimposition, 35 persons were identified. For the remaining 26 persons identification efforts continue. DNA typing was performed at the HLA DQA1 locus and five PM system loci. Results from DNA typing were confirmed by other methods. DNA profiles of family members of 150 missing persons are now being developed using the 6 loci. These DNA profiles will then be compared with those generated from the bone and teeth remains of the unidentified victims.     

Y chromosome polymorphisms in native American and Siberian populations: identification of native American Y chromosome haplotypes

We have initiated a study of ancient male migrations from Siberia to the Americas using Y chromosome polymorphisms. The first polymorphism examined, a C-->T transition at nucleotide position 181 of the DYS199 locus, was previously reported only in Native American populations. To investigate the origin of this DYS199 polymorphism, we screened Y chromosomes from a number of Siberian, Asian, and Native American populations for this and other markers. This survey detected the T allele in all five Native American populations studied at an average frequency of 61%, and in two of nine native Siberian populations, the Siberian Eskimo (21%) and the Chukchi (17%). This finding suggested that the DYS199 T allele may have originated in Beringia and was then spread throughout the New World by the founding populations of the major subgroups of modern Native Americans. We further characterized Native American Y chromosome variation by analyzing two additional Y chromosome polymorphisms, the DYS287 Y Alu polymorphic (YAP) element insertion and a YAP-associated A-->G transition at DYS271, both commonly found in Africans. We found neither African allele associated with the DYS199 T allele in any of the Native American or native Siberian populations. However, we did find DYS287 YAP+ individuals who harbored the DYS199 C allele in one Native American population, the Mixe, and in one Asian group, the Tibetans. A correlation of these Y chromosome alleles in Native Americans with those of the DYS1 locus, as detected by the p49a/p49f (p49a,f) probes on TaqI-digested genomic DNA, revealed a complete association of DYS1 alleles (p49a,f haplotypes) 13, 18, 66, 67 and 69 with the DYS199 T allele, while DYS1 alleles 8 and 63 were associated with both the DYS199 C and T allele.     

Origin and evolution of the 1918 "Spanish" influenza virus hemagglutinin gene

The "Spanish" influenza pandemic killed over 20 million people in 1918 and 1919, making it the worst infectious pandemic in history. Here, we report the complete sequence of the hemagglutinin (HA) gene of the 1918 virus. Influenza RNA for the analysis was isolated from a formalin-fixed, paraffin-embedded lung tissue sample prepared during the autopsy of a victim of the influenza pandemic in 1918. Influenza RNA was also isolated from lung tissue samples from two additional victims of the lethal 1918 influenza: one formalin-fixed, paraffin-embedded sample and one frozen sample obtained by in situ biopsy of the lung of a victim buried in permafrost since 1918. The complete coding sequence of the A/South Carolina/1/18 HA gene was obtained. The HA1 domain sequence was confirmed by using the two additional isolates (A/New York/1/18 and A/Brevig Mission/1/18). The sequences show little variation. Phylogenetic analyses suggest that the 1918 virus HA gene, although more closely related to avian strains than any other mammalian sequence, is mammalian and may have been adapting in humans before 1918.     

Isolation of DNA from the uncultured "Candidatus Liberobacter" species associated with citrus Huanglongbing by RAPD

"Candidatus Liberobacter," the uncultured bacterium associated with citrus Huanglongbing (HLB) disease, is an alpha-Proteobacteria, and two species, "Candidatus L. africanum" and "Candidatus L. asiaticum, " have been characterized by sequence analysis of the 16S rDNA and beta operon (rplKAJL-rpoBC) genes. These genes were isolated by PCR and random cloning of DNA from infected plants. However, this strategy is laborious and allowed selection of only three Liberobacter DNA fragments. In this paper, we described isolation of additional genes using Random Amplified Polymorphic DNA (RAPD). In total, 102 random 10-mer primers were used in PCR reactions on healthy and Liberobacter-infected plant DNA. Eight DNA bands amplified from infected plant DNA were cloned and analyzed. Six of them were found to be part of the Liberobacter genome by sequence and hybridization experiments. On these DNA fragments, four genes were identified: nusG, pgm, omp, and a hypothetical protein gene. These results indicate that RAPD can be used to clone DNA of uncultured organisms.     

DNA fingerprinting analysis by a PCR based method for monitoring the genotoxic effects of heavy metals pollution

Environmental pollutants can have deleterious effects on living organisms. At high concentrations, or at high activities, they can cause acute toxicity damaging cells, tissues and organs. Chronic toxification, on the other hand, can also cause serious damage from bio-accumulation. Plants, as biological indicators, can measure both the actual and the potential effects of pollutants, when they are used to measure effects of heavy metals. We have applied a system of "molecular fingerprinting" based on PCR (RAPD: Random Amplified Polymorphic DNA) to the evaluation of the genotoxic effects of heavy metals in order to estimate the environmental risk connected with their potential mutagenic effects in the model plant Arabidopsis thaliana, ecotype Columbia. Genomic DNA was utilised for RAPD analysis using random primers (10-mers). DNA from plants exposed to heavy metals solution displayed polymorphic bands which were not detectable in DNA of unexposed plants. The enhanced formation of RAPD polymorphisms was also observed in DNA of plant exposed in situ to an industrial pollution source. The comparison between "unexposed" and "exposed" genomes show that RAPD analysis can be used to evaluate how the environmental pollutants modify the structure of DNA in living organisms.     

Isolation, amplification, and sequencing of human mitochondrial DNA obtained from human crab louse, Pthirus pubis (L.), blood meals

The ability to identify individual human hosts based on analyses of blood recovered from the digestive tract of hematophagous arthropods has been a long-term pursuit in both medical and forensic entomology. Blood meal individualization techniques can bring important advancements to studies of vector-borne disease epidemiology. Forensically, these analyses may aid in assailant identification in violent crime cases where blood-feeding insects or their excreta are recovered from victims or at crime scenes. Successful isolation, amplification, and sequencing of human mitochondrial DNA obtained from adult human crab lice fed on human volunteers are reported. Adult lice were removed from recruited volunteers frequenting inner city health clinics. Live lice were killed by freezing and subsequently air dried at ambient temperature. A saliva sample was obtained from each volunteer and served as a DNA reference sample. Volunteers were afforded free, approved pediculosis treatment. Individual lice were subsequently processed using procedures developed for the extraction of mitochondrial DNA from human hair, teeth, and bone. The resulting DNA was amplified by the polymerase chain reaction and sequenced. Our results point to valuable avenues for future entomological research.     

A simple method for extracting DNA from old skeletal material

Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents.     

Short stature and azoospermia in a patient with Y chromosome long arm deletion

We report on a 42-year old male with short stature, azoospermia and a wide deletion of long arm of Y chromosome. On physical examination, the patient showed height of 149 cm (< 1 degree centile) and reduced volume (3 ml) and consistency of the testes. On hormonal evaluation, he showed increased serum gonadotropins and normal serum testosterone levels though its HCG stimulated levels were limited. Serum thyroid hormones were normal. Serum GH levels in baseline evaluation as well as after GHRH and GHRH + pyridostigmine administration were normal. Serum IGF I levels were lower than normal in baseline evaluation whereas its response to the GH administration was in the normal range. The bilateral testicular biopsy showed tubular atrophy, hyalinosis, interstitial sclerosis and a histological picture of a Sertoli cell only syndrome. Moreover the patient showed arthropathy, otopathy, small chin, small mouth and truncal obesity. On genetic evaluation, the patient showed a 46,X,delY (pter--q11.1:) karyotype and loss of several DNA loci on Yq. In fact he preserved short arm SRY, centromeric DYZ3 and more proximal euchromatic region Yq loci, including DYS270, DYS271, DYS272, DYS11, DYS273, DYS274, DYS148, DYS275, and missed more distal DNA loci from DYS246 to DYZ2. These results disclosed a wide Y long arm deletion, including all hypothized Yq azoospermia loci (except for AZFa and probably for one of the RBM genes, which lie proximally to the deletion) and possibly the Y-specific growth control region (GCY), mapped between DYS11 and DYS246 loci. This deletion is responsible for the complete azoospermia of the patient and probably also for his short stature, even if other factors could be implicated in the statural impairment. It further possibly allowed to relate the GCY gene(s) to the control of GH or IGF-I receptor or post-receptor pathway, being the alteration of this gene(s) consistent with the hormonal pattern of the patient.     

Recent developments in the understanding of antinuclear autoantibodies

Cytological identification of soybean mitotic metaphase chromosomes (2n = 40) has been severely limited by their small size and uniform karyomorphology. We have developed fluorescent in situ hybridization (FISH), PCR-primed in situ labelling (PCR-PRINS) procedures, and molecular probes for routine cytological identification and for the physical mapping of soybean somatic chromosomes. Chromosome preparation has been achieved by modifications of previous protocols and through the preparation of root-tip protoplasts prior to chromosome spreading. Initially our probe selection focused on highly repeated DNAs that provide very intense localized hybridization signals. Repetitive gene probes that have proven valuable include the rDNA loci (5S and 45S) which are chromosome specific. We have also developed satellite DNA probes for two different sequence families: the SB92 and the STR120 satellites. Both of these are tandemly arranged at multiple chromosomal loci. By using different cloned examples of each family, we have been able to selectively label unique subsets of soybean chromosomes. Double hybridization with biotin and digoxigenin labeled probes has allowed us to determine the chromosomal overlap between different probes. In addition, we have joined portions of the metaphase chromosome painting patterns with the genetic map by single-copy FISH and PCR-PRINS detection of the RFLP loci G8.15, G17.3, and A199a and A199b. Total genomic DNA in situ hybridization (GISH) patterns were also used to characterize the soybean chromosomes.     

DNA "melting" proteins. I. Effects of bovine pancreatic ribonuclease binding on the conformation and stability of DNA

Bovine pancreatic ribonuclease is a DNA "melting" protein, since it binds with greater overall affinity to the single-stranded than to the double-stranded form of natural and synthetic deoxyribose-containing polynucleotides. As such, the DNA-RNase system provides a simple model for the more complex and biologically relevant melting protein-nucleic acid systems. Aspects of the DNA-RNase interactions which are related to the quantitative assessment of this system as a melting protein model are investigated here. A boundary sedimentation velocity technique is used to measure thermodynamic parameters of the interaction; association constants (Kh and Kc) and site sizes (nh and nc) are determined for the interaction of ribonuclease with native (double helical) and denatured (random coil) DNA. It is shown that log Kh and log Kc are linear functions of log [Na+], binding decreasing with increasing Na+ concentration, with Kh about 2 orders of magnitude smaller than Kc at the ionic strengths studied, nh and nc are approximately 8 and approximately 11 nucleotide residues, respectively, indicating that potential binding sites overlap. Binding to both forms of DNA is non-cooperative. It is shown by CD and ultraviolet spectroscopy that the binding of RNase to single- and double-stranded DNA perturbs the conformations of these polynucleotide conformations very little relative to the unliganded structures. Hydrodynamic methods are used to show that RNase binds to native DNA without altering the overall solution structure of the latter; however conditons which permit binding to, and stabilization of, transiently exposed single-stranded sequences result in a collapse of the stiff native DNA structure. We demonstrate by melting transition studies that ribonuclease does bring about an equilibrium destabilization of native DNA and poly [d(A-T)] and, by applying a ligand-perturbed helic in equilibrium coil theory developed by McGhee (McGhee, J.D. (1976) Biopolymers 15, 1345-1375), it is shown that the extent of the observed destabilization is in semiquantitative accord with expectations based on the measured affinity constants and site sizes for RNase binding to both DNA conformations. Spectral methods are used to show that the relative stability of native DNA sequences of varying base composition is the same in the presence and absence of ribonuclease, strongly arguing that this "melting" ligand "traps" single-stranded sequences transiently exposed by thermal fluctuations. RNase also undergoes an order in equilibrium disorder conformational transition as a function of temperature (the denatured form of RNase stabilizes native DNA, while native RNase destabilizes the native double helix), and the coupled equilibria involved in these interacting conformational changes are interpreted and discussed as possible models of genome regulatory interactions.     

Clinical value of Doppler ultrasound controlled puncture of the inguinal vessels with the "Smart Needle" within the scope of heart catheter examination]

BACKGROUND: Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation (that is, inactivation) of mercaptopurine, azathioprine, and thioguanine and exhibits genetic polymorphism. About 10% of patients have intermediate TPMT activity because of heterozygosity, and about 1 in 300 inherit TPMT deficiency as an autosomal recessive trait. If they receive standard doses of thiopurine medications (for example, 75 mg/m2 body surface area per day), TPMT-deficient patients accumulate excessive thioguanine nucleotides in hematopoietic tissues, which leads to severe and possibly fatal myelosuppression. OBJECTIVE: To elucidate the genetic basis and develop molecular methods for the diagnosis of TPMT deficiency and heterozygosity. DESIGN: Diagnostic test evaluation. SETTING: Research hospital. PATIENTS: The TPMT phenotype was determined in 282 unrelated white persons, and TPMT genotype was determined in all persons who had intermediate TPMT activity (heterozygotes) and a randomly selected, equal number of persons who had high activity. In addition, genotype was determined in 6 TPMT-deficient patients. MEASUREMENTS: Polymerase chain reaction (PCR) assays were developed to detect the G238C transversion in TPMT*2 and the G460A and A719G transitions in TPMT*3 alleles. Radiochemical assay was used to measure TPMT activity. Mutations of TPMT were identified in genomic DNA, and the concordance of TPMT genotype and phenotype was determined. RESULTS: 21 patients who had a heterozygous phenotype were identified (7.4% of sample [95% CI, 4.7% to 11.2%]). TPMT*3A was the most prevalent mutant allele (18 of 21 mutant alleles in heterozygotes; 85%); TPMT*2 and TPMT*3C were more rare (about 5% each). All 6 patients who had TPMT deficiency had two mutant alleles, 20 of 21 patients (95% [CI, 76% to 99.9%]) who had intermediate TPMT activity had one mutant allele, and 21 of 21 patients (100% [CI, 83% to 100%]) who had high activity had no known TPMT mutation. Detection of TPMT mutations in genomic DNA by PCR coincided perfectly with genotypes detected by complementary DNA sequencing. CONCLUSIONS: The major inactivating mutations at the human TPMT locus have been identified and can be reliably detected by PCR-based methods, which show an excellent concordance between genotype and phenotype. The detection of TPMT mutations provides a molecular diagnostic method for prospectively identifying TPMT-deficient and heterozygous patients.     

Observation of the A-DNA to B-DNA transition during unrestrained molecular dynamics in aqueous solution

A large challenge in molecular dynamics (MD) simulations of proteins and nucleic acids is to find the correct "experimental" geometry when a simulation is started a significant distance away from it. In this study, we have carried out four unrestrained approximately 1 ns length MD trajectories in aqueous solution on the DNA duplex d(CCAACGTTGG)2, two beginning in a canonical A-DNA structure and two beginning in a canonical B-DNA structure. As judged by root-mean-squared coordinate deviations, average structures computed from all four of the trajectories converge to within approximately 0.8 to 1.6 angstroms (all atoms) of each other, which is 1.3 to 1.7 angstroms (all atoms of the central six residues from each strand) and 3.1 to 3.6 angstroms (all atoms) away from the B-DNA-like X-ray structure reported for this sequence. To our knowledge, this is the first example of multiple nanosecond molecular dynamics trajectories with full representation of DNA charges, solvent and long range electrostatics that demonstrate both internal consistency (two different starting structures and four different trajectories lead to a consistent average structure) and considerable agreement with the X-ray crystal structure of this sequence and NMR data on duplex DNA in aqueous solution. This internal consistency of structure for a given sequence suggests that one can now begin to realistically examine sequence-dependent structural effects in DNA duplexes using molecular dynamics.     

The expert identification of the remains of the imperial family by means of molecular genetic verification of genealogical relations

The paper presents the results of international complex molecular genetic expert identification of skeletal remains of 9 subjects buried near the Koptiaki road in Ekaterinburg region, presumably belonging to the Romanov Royal Family and persons in their attendance. The armory of methods based on analysis of the least permissible amounts of DNA by the polymerase chain reaction and direct fluorescent sequencing of amplified DNA fragments included the latest scientific technologies. In addition, new methods were developed and used, which have no analogs in the world expert practice. The strategy included identification of biological gender of skeletons, of familial group among exhumed individuals, and of ties of relationship of this family with two independent maternal branches of the Romanov genealogical tree using tracing kindred markers based on mitochondrial DNA (mtDNA). The study was carried out in two stages: in 1992-1993 at Aldermaston Criminology Center of Home Office of the UK with participation of British specialists and in 1995 at Military Medical Institute of Ministry of Defence of the USA in Washington with participation of American specialists. In 1993 five skeletons were identified as Emperor Nicholas II, Empress Alexandra Fedorovna, and their three daughters with 99.6% certainty. From modern criminological viewpoint, the result could not be considered as sufficiently certain for such an extraordinary case, and therefore in 1995 molecular genetic studies of presumable remains of Nicholas II and his brother Prince Georgi? Romanov were carried out again. The results showed absolute positional identity of mtDNA genetic code of these two men due to an extremely rare genetic abnormality (heteroplasmia), and thus the problem of appurtenance of the remains to the Romanov royal family was solved.     

Impact of hepatitis G virus co-infection on the course of hepatitis C virus infection before and after liver transplantation

Large-scale DNA sequencing is creating a sequence infrastructure of great benefit to protein biochemistry. Concurrent with the application of large-scale DNA sequencing to whole genome analysis, mass spectrometry has attained the capability to rapidly, and with remarkable sensitivity, determine weights and amino acid sequences of peptides. Computer algorithms have been developed to use the two different types of data generated by mass spectrometers to search sequence databases. When a protein is digested with a site-specific protease, the molecular weights of the resulting collection of peptides, the mass map or fingerprint, can be determined using mass spectrometry. The molecular weights of the set of peptides derived from the digestion of a protein can then be used to identify the protein. Several different approaches have been developed. Protein identification using peptide mass mapping is an effective technique when studying organisms with completed genomes. A second method is based on the use of data created by tandem mass spectrometers. Tandem mass spectra contain highly specific information in the fragmentation pattern as well as sequence information. This information has been used to search databases of translated protein sequences as well as nucleotide databases such as expressed sequence tag (EST) sequences. The ability to search nucleotide databases is an advantage when analyzing data obtained from organisms whose genomes are not yet completed, but a large amount of expressed gene sequence is available (e.g., human and mouse). Furthermore, a strength of using tandem mass spectra to search databases is the ability to identify proteins present in fairly complex mixtures.     

Race, arousal, attention, exposure and delay: An examination of factors moderating face recognition.

A large percentage of people recently exonerated by DNA evidence were imprisoned on the basis of faulty eyewitness identification. Many of these cases involved victims and suspects of different races. Two studies examined the recognition of Hispanic and Black target faces by Hispanic participants under nonoptimal viewing conditions. When viewing time decreased, recognition performance for same- and other-race faces systematically shifted downward. Recognition accuracy for faces of both races decreased under conditions of high negative arousal and attention load; however, recognition of same-race faces was differentially affected by attention distractors. Face recognition accuracy was not affected by a delay between initial presentation of the faces and the face recognition test. An understanding of how recognition of other-race persons differs from that of same-race persons can assist by reducing misidentifications and ensuring that the perpetrator rather than an innocent person is imprisoned. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A novel chemotherapeutic regimen (interferon alfa, zidovudine, and etretinate) for adult T-cell lymphoma resulting in rapid tumor destruction

A discourse analysis of professional and popular literature describing male violence against women surveyed 165 abstracts and 11 full-length articles. The phrase "male violence" was used only eight times in these writings; male gender was infrequently mentioned, whereas female gender was often present in the identification of victims of male violence. Consequences of the absence of men as perpetrators and visibility of women in the literature on male violence against women are discussed, and change in these discursive practices is called for.     

Isolating the Special Component of Face Recognition: Peripheral Identification and a Mooney Face.

A previous finding argues that, for faces, configural (holistic) processing can operate even in the complete absence of part-based contributions to recognition. Here, this result is confirmed using 2 methods. In both, recognition of inverted faces (parts only) was removed altogether (chance identification of faces in the periphery; no perception of a particularly hard-to-see Mooney face). Recognition of upright faces (configural plus parts), however, remained good. The simplicity of these new "isolation" techniques makes them ideal for (a) assessing configural processing in specialist populations (e.g., children, object experts) and (b) exploring properties of configural processing for faces in detail. As an example of the latter, orientation tuning was tested. Results argue against models in which faces are rotated to upright prior to identification. (PsycINFO Database Record (c) 2010 APA, all rights reserved)