8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations.

BACKGROUND The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination. RESULTS We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes. CONCLUSION MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.

Integrating sequence information in microarray data analysis by free energy modeling

Microarray technology is a high-throughput method for genotyping and gene expression profiling. Limited sensitivity and specificity are one of the essential problems for this technology. Most of existing methods of microarray data analysis have an apparent limitation for they merely deal with the numerical part of microarray data and have made little use of gene sequence information. Because it's the gene sequences that precisely define the physical objects being measured by a microarray, it is natural to make the gene sequences an essential part of the data analysis. This dissertation focused on the development of free energy models to integrate sequence information in microarray data analysis. The models were used to characterize the mechanism of hybridization on microarrays and enhance sensitivity and specificity of microarray measurements. ^ Cross-hybridization is a major obstacle factor for the sensitivity and specificity of microarray measurements. In this dissertation, we evaluated the scope of cross-hybridization problem on short-oligo microarrays. The results showed that cross hybridization on arrays is mostly caused by oligo fragments with a run of 10 to 16 nucleotides complementary to the probes. Furthermore, a free-energy based model was proposed to quantify the amount of cross-hybridization signal on each probe. This model treats cross-hybridization as an integral effect of the interactions between a probe and various off-target oligo fragments. Using public spike-in datasets, the model showed high accuracy in predicting the cross-hybridization signals on those probes whose intended targets are absent in the sample. ^ Several prospective models were proposed to improve Positional Dependent Nearest-Neighbor (PDNN) model for better quantification of gene expression and cross-hybridization. ^ The problem addressed in this dissertation is fundamental to the microarray technology. We expect that this study will help us to understand the detailed mechanism that determines sensitivity and specificity on the microarrays. Consequently, this research will have a wide impact on how microarrays are designed and how the data are interpreted. ^

Distribution of benthic and planktic foraminifera in surface sediments of the Indian and Pakistan continental margin, Arabian Sea

During the Indian Ocean Expedition of the German research vessel "Meteor" and the following cruise with the Pakistani fishing vessel "Machhera" in February and March 1965, sediments were sampled from the shelf, continental slope and the Arabian Basin off Pakistan and India. The biostratigraphic studies are based on sedimentary material from 24 sediment cores up to 480 cm long and 100 grab samples. The faunal residues of the > 160 µ fraction (chiefly foraminifera and pteropods) were determined and counted in order to get an idea of the climatic conditions during the Late Quaternary of this region. Biostratigraphic correlations of these Late Quaternary deposits are only possible if the thanatocoenosis of the surface sediments are well known. The analysis of the benthonic foraminiferal populations resulted in the definition of several foraminiferal facies. The following sequence of forarniniferal facies, named after their most characteristic members, can be distinguished from the shelf to the deep-sea: 1. Ammonia-Florilus facies ; 2. Ammonia-Cancris facies; 3. Cassidulina-Cibicides facies; 4. Uvigerina-Cassidulina facies ; 5. Buliminacea facies ; 6. deepwater facies, partly with Bulimina aculeata or with Nonionidae. On the upper continental slope there is a zone extremely poor in benthonic foraminifera. In this water depth the oxygen minimum layer (0.05-0.02 ml/l) of the water column reaches the slope. Almost no connection can be observed between the living and the dead foraminiferal population of the same sample. The regional distribution of the planktonic foraminifera from plankton tows as well as from the surface sediments shows marked differences in the species composition of faunas from different regions within the area of investigation. That depends on oceanographic conditions such as upwelling, dissolution of carbonate at great depths etc. Based on the results of faunal analysis of samples from the recent sea-floor, a biostratigraphic subdivision of the sediments in the cores was established. The following biostratigraphically defined sections could be distinguished from the top of the sediment cores downwards : 1. Relatively cool climatic conditions are reflected by the foraminifera of the uppermost core sections. 2. The next section is characterized by much warmer conditions (Holocene climatic optimum). The C-14 ages of this interval range from 4000 to 10 000 years B.P. according to different authors. C-14 dates on the material investigated do not give reliable clues. 3. Foraminiferal populations adapted to much colder conditions can be observed in the underlying core section. The boundary between the warm climate reflected by the foraminifera of section 2 and the cold climate (section 3) is relatively sharp. It can be correlated from core to core over the whole area investigated. The cold climate sediments of section 3 are underlain by different cool-, warm- and cold-climate sediments which can only be correlated over very short distances. Since it appears certain that the last really cold conditions ended earlier in the Arabian Sea and its vicinity than in Europe it is recommended not to use the European stratigraphic terms for the Quaternary. Because of the lack of reliable absolute sediment ages for the cores no exact sedimentation rates can be given. According to rough estimates, however, the rates are 1-2 cm/1000 years in the deep basin and up to 40 cm/1000 years on the upper continental slope. Sedimentation rates are always larger near the mouth of the Indus-River than off South India at stations of about the same water depth. Planktonic gastropods (mainly pteropods) cannot be used for biostratigraphic purposes in the region under consideration. All of them seem to be displaced from the shelf. Their distribution there is given in.

Herramienta de apoyo a la docencia en sistemas operativos

El proyecto fin de carrera de herramienta de apoyo a la docencia en Sistemas Operativos quiere ayudar al alumno a entender el funcionamiento de un planificador a corto plazo. Lo hace mediante una representación gráfica de procesos que ocupan o el procesador o distintas unidades de entrada/salida mientras transcurre el tiempo. El tiempo está dividido en ciclos de reloj de un procesador, a lo que a continuación se referirá como unidades de tiempo. Los procesos están definidos por su nombre, la instante de entrada que entran al sistema, su prioridad y la secuencia de unidades de tiempo en el procesador y unidades de entrada/salida que necesitan para terminar su trabajo. El alumno puede configurar el sistema a su gusto en cuanto al número y comportamiento de las unidades de entrada/salida. Puede definir que una unidad solo permita acceso exclusivo a los procesos, es decir que solo un proceso puede ocuparla simultáneamente, o que permita el acceso múltiple a sus recursos. El alumno puede construir un planificador a corto plazo propio, integrarlo en el sistema y ver cómo se comporta. Se debe usar la interfaz Java proporcionada para su construcción. La aplicación muestra datos estadísticos como por ejemplo la eficiencia del sistema (el tiempo activo de la CPU dividido por el tiempo total de la simulación), tiempos de espera de los procesos, etc. Se calcula después de cada unidad de tiempo para que el alumno pueda ver el momento exacto donde la simulación tomó un giro inesperado. La aplicación está compuesta por un motor de simulación que contiene toda la lógica y un conjunto de clases que forman la interfaz gráfica que se presenta al usuario. Estos dos componentes pueden ser reemplazados siempre y cuando se mantenga la definición de sus conectores igual. La aplicación la he hecho de manejo muy simple e interfaz fácil de comprender para que el alumno pueda dedicar todo su tiempo a probar distintas configuraciones y situaciones y así entender mejor la asignatura. ABSTRACT. The project is called “Tool to Support Teaching of the Subject Operating Systems” and is an application that aims to help students understand on a deeper level the inner workings of how an operating system handles multiple processes in need of CPU time by the means of a short-term planning algorithm. It does so with a graphical representation of the processes that occupy the CPU and different input/output devices as time passes by. Time is divided in CPU cycles, from now on referred to as time units. The processes are defined by their name, the moment they enter the system, their priority and the sequence of time units they need to finish their job. The student can configure the system by changing the number and behavior of the input/output devices. He or she can define whether a device should only allow exclusive access, i.e. only one process can occupy it at any given time, or if it should allow multiple processes to access its resources. The student can build a planning algorithm of his or her own and easily integrate it into the system to see how it behaves. The provided Java interface and the programming language Java should be used to build it. The application shows statistical data, e.g. the efficiency of the system (active CPU time divided by total simulation time) and time spent by the processes waiting in queues. The data are calculated after passing each time unit in order for the student to see the exact moment where the simulation took an unexpected turn. The application is comprised of a simulation motor, which handles all the logic, and a set of classes, which is the graphical user interface. These two parts can be replaced individually if the definition of the connecting interfaces stays the same. I have made the application to be very easy to use and with an easy to understand user interface so the student can spend all of his or her time trying out different configurations and scenarios in order to understand the subject better.

Sequence-specific polypeptoids: A diverse family of heteropolymers with stable secondary structure

We have synthesized and characterized a family of structured oligo-N-substituted-glycines (peptoids) up to 36 residues in length by using an efficient solid-phase protocol to incorporate chemically diverse side chains in a sequence-specific fashion. We investigated polypeptoids containing side chains with a chiral center adjacent to the main chain nitrogen. Some of these sequences have stable secondary structure, despite the achirality of the polymer backbone and its lack of hydrogen bond donors. In both aqueous and organic solvents, peptoid oligomers as short as five residues give rise to CD spectra that strongly resemble those of peptide α-helices. Differential scanning calorimetry and CD measurements show that polypeptoid secondary structure is highly stable and that unfolding is reversible and cooperative. Thermodynamic parameters obtained for unfolding are similar to those obtained for the α-helix to coil transitions of peptides. This class of biomimetic polymers may enable the design of self-assembling macromolecules with novel structures and functions.

Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence

Unique, small sequences (sequence tag sites) have been identified at the 3′ ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3′ unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3′ hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3′ end sequence to various Alu positions as much as 600 kb upstream. These YACs were retrofitted with a NeoR and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.

Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs

Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA. B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immune-stimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA. In type 12 genomes, the distribution of CpG-flanking bases is similar to that predicted by chance. However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15- to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5′ side or a G on the 3′ side. Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs. Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing. These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.

Movements of truncated kinesin fragments with a short or an artificial flexible neck

To investigate the role of the neck domain of kinesin, we used optical trapping nanometry to perform high-resolution measurements of the movements and forces produced by recombinant kinesin fragments in which the neck domains were shortened or replaced by an artificial random coil. Truncated kinesin fragments (K351) that contain a motor domain consisting of ≈340 aa and a short neck domain consisting of ≈11 aa showed fast movement (800 nm/s) and 8-nm steps. Such behavior was similar to that of recombinant fragments containing the full-length neck domain (K411) and to that of native kinesin. Kinesin fragments lacking the short neck domain (K340), however, showed very slow movement (<50 nm/s), as previously reported. Joining an artificial 11-aa sequence that was expected to form a flexible random chain to the motor domain (K340–chain) produced normal fast (≈700 nm/s) and stepwise movement. The results suggest that the neck domain does not act as a rigid lever arm to magnify the structural change at the catalytic domain as has been believed for myosin, but it does act as a flexible joint to guarantee the mobility of the motor domain.

Semaphorin 3A growth cone collapse requires a sequence homologous to tarantula hanatoxin

Axonal guidance is key to the formation of neuronal circuitry. Semaphorin 3A (Sema 3A; previously known as semaphorin III, semaphorin D, and collapsin-1), a secreted subtype of the semaphorin family, is an important axonal guidance molecule in vitro and in vivo. The molecular mechanisms of the repellent activity of semaphorins are, however, poorly understood. We have now found that the secreted semaphorins contain a short sequence of high homology to hanatoxin, a tarantula K+ and Ca2+ ion channel blocker. Point mutations in the hanatoxin-like sequence of Sema 3A reduce its capacity to repel embryonic dorsal root ganglion axons. Sema 3A growth cone collapse activity is inhibited by hanatoxin, general Ca2+ channel blockers, a reduction in extracellular or intracellular Ca2+, and a calmodulin inhibitor, but not by K+ channel blockers. Our data support an important role for Ca2+ in mediating the Sema 3A response and suggest that Sema 3A may produce its effects by causing the opening of Ca2+ channels.

Sequence variation in the Fanconi anemia gene FAA

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (FAA) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for FAA. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the FAA gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115–1118del and 3788–3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788–3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that FAA is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.

Assembly of τ protein into Alzheimer paired helical filaments depends on a local sequence motif (306VQIVYK311) forming β structure

We have searched for a minimal interaction motif in τ protein that supports the aggregation into Alzheimer-like paired helical filaments. Digestion of the repeat domain with different proteases yields a GluC-induced fragment comprising 43 residues (termed PHF43), which represents the third repeat of τ plus some flanking residues. This fragment self assembles readily into thin filaments without a paired helical appearance, but these filaments are highly competent to nucleate bona fide PHFs from full-length τ. Probing the interactions of PHF43 with overlapping peptides derived from the full τ sequence yields a minimal hexapeptide interaction motif of 306VQIVYK311 at the beginning of the third internal repeat. This motif coincides with the highest predicted β-structure potential in τ. CD and Fourier transform infrared spectroscopy shows that PHF43 acquires pronounced β structure in conditions of self assembly. Point mutations in the hexapeptide region by proline-scanning mutagenesis prevent the aggregation. The data indicate that PHF assembly is initiated by a short fragment containing the minimal interaction motif forming a local β structure embedded in a largely random-coil protein.

Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5′ leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reassayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the design of vectors for biotechnology and gene therapy.