ABSTRACT
BACKGROUND/AIM: Nucleic acid metabolism is biochemically compartmentalized to the nucleus. Thus, it is necessary to define the proteome of the various macromolecular structures within this organelle. MATERIALS AND METHODS: We isolated the nuclear matrix (NM) fraction from rat liver by sequential centrifugation steps at 13,000 rpm, staggered between endogenous nuclease treatment for 2 h at 37°C, followed by high-salt (H.S.; 2.0 M NaCl) and non-ionic detergent extractions (0.1%- or 1.0% Triton X-100) to eliminate the bulk of chromosomal DNA/RNA, histone proteins and the nuclear envelope (NE). RESULTS: Integrity of the NM and NE structures was confirmed by electron microscopy. Next, we analyzed the NM proteome on a 20% polyacrylamide gel using the PhastSystem. We observed the absence of histone proteins and the characteristic presence of the lamins by Coomassie blue staining. By contrast, upon silver staining, following electrophoretic separation with a Tris-Borate-EDTA buffer, we observed the NM-associated nucleic RNA and protein-free ADP-ribose polymers. While polymers are found in much lower concentration than RNA in NM, they were purified by affinity chromatography on boronate resin prior to electrophoresis. We observed the electrophoretic resolution of free ADP-ribose chains (5-25 units) by silver staining. CONCLUSION: The significance of our observations to cancer studies and carcinogenesis is discussed.
Subject(s)
Neoplasms/chemistry , Nuclear Envelope/chemistry , Nuclear Matrix-Associated Proteins/chemistry , Nuclear Matrix/chemistry , Poly(ADP-ribose) Polymerases/chemistry , Proteome/chemistry , Animals , Cell Nucleolus/chemistry , Cell Nucleolus/metabolism , Electrophoresis/methods , Neoplasms/metabolism , Nuclear Envelope/metabolism , Nuclear Matrix/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Proteome/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The present study characterizes the genetic variability of Mulatto population based on the polymorphism of six miniSTR autosomal loci, known as Non Codis 01 and 02 (NC01 and NC02) and evaluate their applicability in forensic genetics. A sample of 102 unrelated Brazilian mulattoes were genotyped for miniSTR loci D1S1677, D2S441, D4S2364 (miniplex NC02) and 45 individuals for D10S1248, D14S1434, D22S1045 (miniplex NC01). No significant deviations from Hardy-Weinberg equilibrium expectations were detected. The combined power of discrimination (PD) and mean power of exclusion (PE) were 0.999996 and 0.98991, respectively. The results also support the effectiveness of the NC01and NC02 miniplexes for human identification.
Subject(s)
Ethnicity/genetics , Genetic Variation , Genetics, Population , Microsatellite Repeats , Brazil , DNA Fingerprinting , Gene Frequency , Genotype , HumansSubject(s)
Genetics, Population , Tandem Repeat Sequences , DNA Fingerprinting , Gene Frequency , Humans , Libya , Polymerase Chain ReactionABSTRACT
Three sampled populations of unrelated males--African American, Caucasian, and Hispanic, all from Texas-were typed for 16 Y short tandem repeat (STR) markers using the AmpFlSTR Yfiler kit. These samples also were typed previously for the 13 core CODIS autosomal STR loci. Most of the 16 marker haplotypes (2478 out of 2551 distinct haplotypes) were observed only once in the data sets. Haplotype diversities were 99.88%, 99.89%, and 99.87% for the African American, Caucasian, and Hispanic sample populations, respectively. F(ST) values were very small when a haplotype comprised 10-16 markers. This suggests that inclusion of substructure correction is not required. However, haplotypes consisting of fewer loci may require the inclusion of F(ST) corrections. The testing of independence of autosomal and Y STRs supports the proposition that the frequencies of autosomal and Y STR profiles can be combined using the product rule.
Subject(s)
Chromosomes, Human, Y , DNA Fingerprinting , Haplotypes , Racial Groups/genetics , Tandem Repeat Sequences , Genetic Markers , Genetic Variation , Humans , Male , Polymerase Chain Reaction , TexasABSTRACT
Father-son pairs from three populations (African American, Caucasian, and Hispanic) of Texas were typed for the 17 Y STR markers DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS456, DYS458, DYS635, DYS448, and Y GATA H4 using the AmpFlSTR YfilerTM kit. With 49,578 allele transfers, 102 mutations were detected. One three-step and four two-step mutations were found, and all others (95.1%) were one-step mutations. The number of gains (48) and losses (54) of repeats were nearly similar. The average mutation rate in the total population is 2.1 x 10(-3) per locus (95% CI (1.7-2.5)x10(-3)). African Americans showed a higher mutation rate (3.0 x 10(-3); 95% CI (2.4-4.0)x10(-3)) than the Caucasians (1.7 x 10(-3); 95% CI (1.1-2.5)x10(-3)) and Hispanics (1.5 x 10(-3); 95% CI (1.0-2.2)x10(-3)), but grouped by repeat-lengths, such differences were not significant. Mutation is correlated with relative length of alleles, i.e., longer alleles are more likely to mutate compared with the shorter ones at the same locus. Mutation rates are also correlated with the absolute number of repeats, namely, alleles with higher number of repeats are more likely to mutate than the shorter ones (p-value=0.030). Finally, occurrences of none, one, and two mutations over the father-son transmission of alleles were consistent with the assumption of independence of mutation rates across loci.
Subject(s)
Chromosomes, Human, Y , Genetics, Population , Microsatellite Repeats , Mutation , Population Groups/genetics , Black or African American , DNA Fingerprinting/methods , Fathers , Hispanic or Latino , Humans , Male , Nuclear Family , Texas , White PeopleABSTRACT
Null alleles can occur with any PCR-based STR typing system. They generally are due to deletions within the target region or primer binding sites or by primer binding site mutations that destabilize hybridization of at least one of the primers flanking the target region. Although not common, null types were detected at the DYS448 locus in seven out of 1,005 unrelated males in the Hispanic population. Of these DYS448 null types, four individuals displayed an apparent duplication at the DYS437 locus. The additional allele observed at the DYS437 locus is in actuality a smaller-sized DYS448 amplicon, which is the result of a deletion of the invariant N42 base pair domain and downstream repeats within the DYS448 locus. Thus, some DYS448 null types are not truly null. A true DYS448 null allele carried numerous primer binding site variants and a large deletion including the N42 base pair domain and surrounding or downstream repeat regions. The presence of null alleles is not a real concern for interpretation of Y STR loci evidence; current methods for interpreting Y STR profiles easily accommodate such phenomena.
Subject(s)
Alleles , Chromosomes, Human, Y , DNA Fingerprinting , Tandem Repeat Sequences , Haplotypes , Humans , Male , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.
Subject(s)
Chromosomes, Human, Y , DNA Fingerprinting/standards , Polymerase Chain Reaction/standards , Sex Determination Processes , Tandem Repeat Sequences , Animals , DNA Primers , Female , Genetic Markers , Genotype , Humans , Male , Polymerase Chain Reaction/methods , Reproducibility of Results , Sensitivity and Specificity , TemperatureABSTRACT
A total of 2443 male individuals, previously typed for the 13 CODIS STR loci, distributed across the five North American population groups African American, Asian, Caucasian, Hispanic, and Native American were typed for the Y-STR loci DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439 using the PowerPlex Y System. All population samples were highly polymorphic for the 12 Y-STR loci with the marker DYS385a/b being the most polymorphic across all sample populations. The Native American population groups demonstrated the lowest genetic diversity, most notably at the DYS393 and DYS437 loci. Almost all of the 12-locus haplotypes observed in the sample populations were represented only once in the database. Haplotype diversities were greater than 99.6% for the African Americans, Caucasians, Hispanics, and Asians. The Native Americans had the lowest haplotype diversities (Apaches, 97.0%; Navajo, 98.1%). Population substructure effects were greater for Y-haplotypes, compared with that for the autosomal loci. For the apportionment of variance for the 12 Y-STRs, the within sample population variation was the largest component (>98% for each major population group and approximately 97% in Native Americans), and the variance component contributed by the major population groups was less than the individual component, but much greater than among sample populations within a major group (11.79% versus 1.02% for African Americans/Caucasians/Hispanics and 15.35% versus 1.25% for all five major populations). When each major population is analyzed individually, the R(ST) values were low but showed significant among group heterogeneity. In 692 confirmed father-son pairs, 14 mutation events were observed with the average rate of 1.57x10(-3)/locus/generation (a 95% confidence bound of 0.83x10(-3) to 2.69x10(-3)). Since the Y-STR loci reside on the non-recombining region of the Y chromosome, the counting method is one approach suggested for conveying an estimate of the rarity of the Y-haplotype. Because the Y-STR loci are not all in disequilibrium to the same extent, the counting method is a very conservative approach. The data also support that autosomal STR frequencies can be multiplied by the upper bound frequency estimate of a Y-haplotype in the individual population group or those pooled into major population groups (i.e., Caucasian, African American, Hispanic, and Asian). These analyses support use of the haplotype population data for estimating Y-STR profile frequencies for populations residing in North America.
Subject(s)
Chromosomes, Human, Y/genetics , Haplotypes , Population Groups/genetics , Tandem Repeat Sequences , Canada , Genetics, Population , Humans , United StatesABSTRACT
Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.