Using a pericentromeric interspersed repeat to recapitulate the phylogeny and expansion of human centromeric segmental duplications

Despite considerable advances in sequencing of the human genome over the past few years, the organization and evolution of human pericentromeric regions have been difficult to resolve. This is due, in part, to the presence of large, complex blocks of duplicated genomic sequence at the boundary between centromeric satellite and unique euchromatic DNA. Here, we report the identification and characterization of an approximately 49-kb repeat sequence that exists in more than 40 copies within the human genome. This repeat is specific to highly duplicated pericentromeric regions with multiple copies distributed in an interspersed fashion among a subset of human chromosomes. Using this interspersed repeat (termed PIR4) as a marker of pericentromeric DNA, we recovered and sequence-tagged 3 Mb of pericentromeric DNA from a variety of human chromosomes as well as nonhuman primate genomes. A global evolutionary reconstruction of the dispersal of PIR4 sequence and analysis of flanking sequence supports a model in which pericentromeric duplications initiated before the separation of the great ape species (>12 MYA). Further, analyses of this duplication and associated flanking duplications narrow the major burst of pericentromeric duplication activity to a time just before the divergence of the African great ape and human species (5 to 7 MYA). These recent duplication exchange events substantially restructured the pericentromeric regions of hominoid chromosomes and created an architecture where large blocks of sequence are shared among nonhomologous chromosomes. This report provides the first global view of the series of historical events that have reshaped human pericentromeric regions over recent evolutionary time.     

The protein kinase C beta II exon confers mRNA instability in the presence of high glucose concentrations

Previous studies showed that short term exposure of cells to high glucose destabilized protein kinase C (PKC) betaII mRNA, whereas PKCbetaI mRNA levels remained unaltered. Because PKCbeta mRNAs share common sequences other than the PKCbetaII exon encoding a different carboxyl terminus, we examined PKCbetaII mRNA for a cis-acting region that could confer glucose-induced destabilization. A beta-globin/growth hormone reporter con struct containing the PKCbetaII exon was transfected into human aorta and rat vascular smooth muscle cells (A10) to follow glucose-induced destabilization. Glucose (25 mm) exposure destabilized PKCbetaII chimeric mRNA but not control mRNA. Deletion analysis and electrophoretic mobility shift assays followed by UV cross-linking experiments demonstrated that a region introduced by inclusion of the betaII exon was required to confer destabilization. Although a cis-acting element mapped to 38 nucleotides within the betaII exon was necessary to bestow destabilization, it was not sufficient by itself to confer complete mRNA destabilization. Yet, in intact cells antisense oligonucleotides complementary to this region blocked glucose-induced destabilization. These results suggest that this region must function in context with other sequence elements created by exon inclusion involved in affecting mRNA stability. In summary, inclusion of an exon that encodes PKCbetaII mRNA introduces a cis-acting region that confers destabilization to the mRNA in response to glucose.     

Accuracy and repeatability of a pressure measurement system in the patellofemoral joint

The objective of this study was to assess how accurately and repeatably the Iscan system measures force and pressure in the natural patellofemoral joint. These measurements must be made to test widely held assumptions about the relationships between mechanics, pain and cartilage degeneration. We assessed the system's accuracy by using test rigs in a materials testing machine to apply known forces and force distributions across the sensor. The root mean squared error in measuring resultant force (for five trials at each of seven load levels) was 6.5±4.4% (mean±standard deviation over all trials at all load levels), while the absolute error was −5.5±5.6%. For force distribution, the root mean squared error (for five trials at each of five force distributions) was 0.86±0.58%, while the absolute error was −0.22±1.03%. We assessed the repeatability of the system's measurements of patellofemoral contact force, pressure and force distribution in four cadaver specimens loaded in continuous and static flexion. Variability in measurement (standard deviation expressed as a percentage of the mean) was 9.1% for resultant force measurements and 3.0% for force distribution measurements for static loads, and 7.3% for resultant force and 2.2% for force distribution measurements for continuous flexion. Cementing the sensor to the cartilage lowered readings of resultant force by 31±32% (mean±standard deviation), area by 24±13% and mean pressure by 9±34% (relative to the uncemented sensor). Maximum pressure measurement, however, was 24±43% higher in the cemented sensor than in the uncemented sensor. The results suggest that the sensor measures force distribution more accurately and repeatably than absolute force. A limitation of our work, however, is that the sensor must be cemented to the patellar articular surface to make the force distribution measurements, and our results suggest that this process reduces the accuracy of force, pressure and area measurements. Our results suggest that the Iscan system's pressure measurement accuracy and repeatability are comparable to that of Fuji Prescale film, but its advantages are that it is thinner than most Fuji Prescale film, it measures contact area more accurately and that it makes continuous measurements of force, pressure and area.     

Gene replacement and quantitative mass spectrometry approaches validate guanosine monophosphate synthetase as essential for Mycobacterium tuberculosis growth

Guanosine monophosphate synthetase (GMPS), encoded by guaA gene, is a key enzyme for guanine nucleotide biosynthesis in Mycobacterium tuberculosis. The guaA gene from several bacterial pathogens has been shown to be involved in virulence; however, no information about the physiological effect of direct guaA deletion in M. tuberculosis has been described so far. Here, we demonstrated that the guaA gene is essential for M. tuberculosis H37Rv growth. The lethal phenotype of guaA gene disruption was avoided by insertion of a copy of the ortholog gene from Mycobacterium smegmatis, indicating that this GMPS protein is functional in M. tuberculosis. Protein validation of the guaA essentiality observed by PCR was approached by shotgun proteomic analysis. A quantitative method was performed to evaluate protein expression levels, and to check the origin of common and unique peptides from M. tuberculosis and M. smegmatis GMPS proteins. These results validate GMPS as a molecular target for drug design against M. tuberculosis, and GMPS inhibitors might prove to be useful for future development of new drugs to treat human tuberculosis.     

The role of unequal crossover in alpha-satellite DNA evolution: a computational analysis.

Human DNA consists of a large number of tandem repeat sequences. Such sequences are usually called satellites, with the primary example being the centromeric alpha-satellite DNA. The basic repeat unit of the alpha-satellite DNA is a 171 bp monomer. Arbitrary monomer pairs usually have considerable sequence divergence (20-40%). However, with the exception of peripheral alpha-satellite DNA, monomers can be grouped into blocks of k-monomers (4 < or = k < or = 20) between which the divergence rate is much smaller (e.g., 5%). Perhaps the simplest and best understood mechanism for tandem repeat array evolution is unequal crossover. Although it is possible that alpha-satellite sequences developed as a result of subsequent unequal crossovers only, no formal computational framework seems to have been developed to verify this possibility. In this paper, we develop such a framework and report on experiments which imply that pericentromeric alpha-satellite segments (which are devoid of higher order structure) are evolutionarily distinct from the higher order repeat segments. It is likely that the higher order repeats developed independently in distinct regions of the genome and were carried into their current locations through an unknown mechanism of transposition.