Bos taurus genome assembly.

BACKGROUND: We present here the assembly of the bovine genome. The assembly method combines the BAC plus WGS local assembly used for the rat and sea urchin with the whole genome shotgun (WGS) only assembly used for many other animal genomes including the rhesus macaque. RESULTS: The assembly process consisted of multiple phases: First, BACs were assembled with BAC generated sequence, then subsequently in combination with the individual overlapping WGS reads. Different assembly parameters were tested to separately optimize the performance for each BAC assembly of the BAC and WGS reads. In parallel, a second assembly was produced using only the WGS sequences and a global whole genome assembly method. The two assemblies were combined to create a more complete genome representation that retained the high quality BAC-based local assembly information, but with gaps between BACs filled in with the WGS-only assembly. Finally, the entire assembly was placed on chromosomes using the available map information.Over 90% of the assembly is now placed on chromosomes. The estimated genome size is 2.87 Gb which represents a high degree of completeness, with 95% of the available EST sequences found in assembled contigs. The quality of the assembly was evaluated by comparison to 73 finished BACs, where the draft assembly covers between 92.5 and 100% (average 98.5%) of the finished BACs. The assembly contigs and scaffolds align linearly to the finished BACs, suggesting that misassemblies are rare. Genotyping and genetic mapping of 17,482 SNPs revealed that more than 99.2% were correctly positioned within the Btau_4.0 assembly, confirming the accuracy of the assembly. CONCLUSION: The biological analysis of this bovine genome assembly is being published, and the sequence data is available to support future bovine research.

The complete genome of an individual by massively parallel DNA sequencing.

The association of genetic variation with disease and drug response, and improvements in nucleic acid technologies, have given great optimism for the impact of 'genomic medicine'. However, the formidable size of the diploid human genome, approximately 6 gigabases, has prevented the routine application of sequencing methods to deciphering complete individual human genomes. To realize the full potential of genomics for human health, this limitation must be overcome. Here we report the DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels. This sequence was completed in two months at approximately one-hundredth of the cost of traditional capillary electrophoresis methods. Comparison of the sequence to the reference genome led to the identification of 3.3 million single nucleotide polymorphisms, of which 10,654 cause amino-acid substitution within the coding sequence. In addition, we accurately identified small-scale (2-40,000 base pair (bp)) insertion and deletion polymorphism as well as copy number variation resulting in the large-scale gain and loss of chromosomal segments ranging from 26,000 to 1.5 million base pairs. Overall, these results agree well with recent results of sequencing of a single individual by traditional methods. However, in addition to being faster and significantly less expensive, this sequencing technology avoids the arbitrary loss of genomic sequences inherent in random shotgun sequencing by bacterial cloning because it amplifies DNA in a cell-free system. As a result, we further demonstrate the acquisition of novel human sequence, including novel genes not previously identified by traditional genomic sequencing. This is the first genome sequenced by next-generation technologies. Therefore it is a pilot for the future challenges of 'personalized genome sequencing'.

Electrostatic steering at acetylcholine binding sites.

The electrostatic environments near the acetylcholine binding sites on the nicotinic acetylcholine receptor (nAChR) and acetylcholinesterase were measured by diffusion-enhanced fluorescence energy transfer (DEFET) to determine the influence of long-range electrostatic interactions on ligand binding kinetics and net binding energy. Changes in DEFET from variously charged Tb3+ -chelates revealed net potentials of -20 mV at the nAChR agonist sites and -14 mV at the entrance to the AChE active site, in physiological ionic strength conditions. The potential at the alphadelta-binding site of the nAChR was determined independently in the presence of d-tubocurarine to be -14 mV; the calculated potential at the alphagamma-site was approximately threefold stronger than at the alphadelta-site. By determining the local potential in increasing ionic strength, Debye-Hückel theory predicted that the potentials near the nAChR agonist binding sites are constituted by one to three charges in close proximity to the binding site. Examination of the binding kinetics of the fluorescent acetylcholine analog dansyl-C6-choline at ionic strengths from 12.5 to 400 mM revealed a twofold decrease in association rate. Debye-Hückel analysis of the kinetics revealed a similar charge distribution as seen by changes in the potentials. To determine whether the experimentally determined potentials are reflected by continuum electrostatics calculations, solutions to the nonlinear Poisson-Boltzmann equation were used to compute the potentials expected from DEFET measurements from high-resolution models of the nAChR and AChE. These calculations are in good agreement with the DEFET measurements for AChE and for the alphagamma-site of the nAChR. We conclude that long-range electrostatic interactions contribute -0.3 and -1 kcal/mol to the binding energy at the nAChR alphadelta- and alphagamma-sites due to an increase in association rates.

The DNA sequence, annotation and analysis of human chromosome 3.

After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.

The finished DNA sequence of human chromosome 12.

Human chromosome 12 contains more than 1,400 coding genes and 487 loci that have been directly implicated in human disease. The q arm of chromosome 12 contains one of the largest blocks of linkage disequilibrium found in the human genome. Here we present the finished sequence of human chromosome 12, which has been finished to high quality and spans approximately 132 megabases, representing approximately 4.5% of the human genome. Alignment of the human chromosome 12 sequence across vertebrates reveals the origin of individual segments in chicken, and a unique history of rearrangement through rodent and primate lineages. The rate of base substitutions in recent evolutionary history shows an overall slowing in hominids compared with primates and rodents.

The Atlas genome assembly system.

Atlas is a suite of programs developed for assembly of genomes by a "combined approach" that uses DNA sequence reads from both BACs and whole-genome shotgun (WGS) libraries. The BAC clones afford advantages of localized assembly with reduced computational load, and provide a robust method for dealing with repeated sequences. Inclusion of WGS sequences facilitates use of different clone insert sizes and reduces data production costs. A core function of Atlas software is recruitment of WGS sequences into appropriate BACs based on sequence overlaps. Because construction of consensus sequences is from local assembly of these reads, only small (<0.1%) units of the genome are assembled at a time. Once assembled, each BAC is used to derive a genomic layout. This "sequence-based" growth of the genome map has greater precision than with non-sequence-based methods. Use of BACs allows correction of artifacts due to repeats at each stage of the process. This is aided by ancillary data such as BAC fingerprint, other genomic maps, and syntenic relations with other genomes. Atlas was used to assemble a draft DNA sequence of the rat genome; its major components including overlapper and split-scaffold are also being used in pure WGS projects.

Site-selective agonist binding to the nicotinic acetylcholine receptor from Torpedo californica.

Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites. Equilibrium binding measurements show that the alpha gamma site has a lower fluorescence than the alpha delta site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5-10-fold faster from the alpha gamma site than from the alpha delta site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the alpha delta site (K = 3 nM) than to the alpha gamma site (K = 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.

[Analysis of caspases activity of hematopoietic progenitor cells and its significance in myelodysplastic syndromes].

OBJECTIVE: To investigate the changes of apoptosis and the activity of caspases 3 and 9 in bone marrow hematopoietic progenitor cells in myelodysplastic syndromes (MDS). METHODS: Bone marrow hematopoietic progenitor cells (including both CD(34)(+) and CD(34)(-) cells) were collected by negative selection in 34 patients with MDS. Apoptosis was measured with Annexin V assay and activities of caspases 3 and 9 by spectrophotometer. RESULTS: 1. Apoptosis was significantly increased in MDS-RA (39.5%, P < 0.01) and MDS-RAEB (31.0%, P < 0.05), but was not different statistically in MDS-RAEBt/AML (18.8%) compared with that of control. 2. Activities of caspases 3 and 9 increased 45 and 20 fold in MDS-RA, increased 14 and 2 fold in MDS-RAEB, respectively and was not increased in MDS-RAEBt/AML compared with that of control. 3. Apoptosis and activities of caspases 3 and 9 reduced in 3 cases of MDS-RAEB group who progressed into AML. CONCLUSION: Increased activities of caspases 3 and 9 may be one of causes of excessive apoptosis in MDS. With progress to AML, activities of caspases 3 and 9 and apoptosis reduced. Reduced activity of caspase 9 may result in apoptosis "escape" and progression into AML.

Substituent-Induced Perturbation Symmetries and Distortions of meso-tert-Butylporphyrins.

The out-of-plane and in-plane distortions of a series of nickel(II) meso-substituted porphyrins with 0, 1, 2, or 4 tert-butyl groups [nickel(II) porphine (NiP), nickel(II) mono-tert-butylporphyrin (NiMtBuP), nickel(II) di-tert-butylporphyrin (NiDtBuP), and nickel(II) tetra-tert-butylporphyrin (NiTtBuP)] are investigated using molecular mechanics (MM) calculations, X-ray crystallography, UV-visible absorption spectroscopy, and resonance Raman spectroscopy. MM calculations are used to predict the stable conformations for this series of porphyrins. The out-of-plane distortions are then analyzed in terms of displacements along the normal coordinates of the porphyrin macrocycle using a new normal-coordinate structural decomposition method. As expected, the distortions are found to occur primarily along the lowest-frequency normal coordinate of each symmetry type and the distortions could be adequately simulated using only the lowest-frequency normal coordinates as a basis (the minimal basis). However, the distortions could be simulated significantly more accurately by extending the minimal basis by including the second-lowest-frequency normal coordinate of all symmetries. Using the extended basis is most important for the in-plane distortions. Detailed analysis of the types of distortion revealed that both the out-of-plane and the in-plane distortions depend on the perturbation symmetry of the peripheral substituents. The symmetry primarily depends on the pattern of substitution (number and positions of substituents) and the orientations of substituents. Often the perturbation symmetry can be predicted for a given porphyrin simply from the possible orientations of the substituents. Then, the main type(s) of symmetric deformation occurring for each possible molecular symmetry can be readily predicted from a D(4)(h)() correlation table. The stable conformers predicted by MM for the series of tert-butyl-substituted porphyrins confirm this simple but informative approach. Experimental verification of the calculated contributions of the symmetric deformations is provided by normal-coordinate structural decomposition of the available X-ray crystal structures of NiP, NiMtBuP, and NiDtBuP. The solid-state results are also supported by the resonance Raman and UV-visible absorption spectroscopic characterization of the porphyrins in solutions. The X-ray crystal structure of NiMtBuP is reported here for the first time.

Axial Coordination and Conformational Heterogeneity of Nickel(II) Tetraphenylporphyrin Complexes with Nitrogenous Bases.

Axial ligation of nickel(II) 5,10,15,20-tetraphenylporphyrin (NiTPP) with pyrrolidine or piperidine has been investigated using X-ray crystallography, UV-visible spectroscopy, resonance Raman spectroscopy, and molecular mechanics (MM) calculations. By varying the pyrrolidine concentration in dichloromethane, distinct nu(4) Raman lines are found for the four-, five-, and six-coordinate species of NiTPP. The equilibrium constants for addition of the first and second pyrrolidine axial ligands are 1.1 and 3.8 M(-)(1), respectively. The axial ligands and their orientations influence the type and magnitude of the calculated nonplanar distortion. The differences in the calculated energies of the conformers having different ligand rotational angles are small so they may coexist in solution. Because of the similarity in macrocyclic structural parameters of these conformers and the free rotation of the axial ligands, narrow and symmetric nu(2) and nu(8) Raman lines are observed. Nonetheless, the normal-coordinate structural-decomposition analysis of the nonplanar distortions of the calculated structures and the crystal structure of the bis(piperidine) complex reveals a relationship between the orientations of axial ligand(s) and the macrocyclic distortions. For the five-coordinate complex with the plane of the axial ligand bisecting the Ni-N(pyrrole) bonds, a primarily ruffled deformation results. With the ligand plane eclipsing the Ni-N(pyrrole) bonds, a mainly saddled deformation occurs. With the addition of the second axial ligand, the small doming of the five-coordinate complexes disappears, and ruffling or saddling deformations change depending on the relative orientation of the two axial ligands. The crystal structure of the NiTPP bis(piperidine) complex shows a macrocycle distortion composed of wav(x) and wav(y) symmetric deformations, but no ruffling, saddling, or doming. The difference in the calculated and observed distortions results partly from the phenyl group orientation imposed by crystal packing forces. MM calculations predict three stable conformers (ruf, sad, and planar) for four-coordinate NiTPP, and resonance Raman evidence for these conformers was given previously.