Accurate whole human genome sequencing using reversible terminator chemistry

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.     

Cell lines producing human T-cell lymphoma virus show altered HLA expression

Human T-cell leukaemia/lymphoma virus (HTLV) can be identified in fresh and cultured T-lymphocytes from patients with adult T-cell malignancies. HLA typing of the peripheral blood lymphocytes and cultured cell lines from the patient from which the virus was originally isolated suggested the expression of additional HLA-A and -B locus antigens on the HTLV positive cultured T-cells that were not present on the EBV transformed B-cell line or on the peripheral blood lymphocytes. Peripheral blood lymphocytes (PBLs) and T-cell lines established from patients and cord blood lymphocytes, infected with virus by co-culture with T-cell lines, were typed for HLA antigens with alloantisera and in addition tested for reactivity with a monoclonal antibody (4D12) which recognizes a polymorphic HLA class-I antigen. In all HTLV positive cells, with demonstrable provirus replication, altered HLA alloantigen expression was observed. This may be explained by the observations reported in the accompanying paper which shows homology between the envelope gene region of HTLV and the region of an HLA-B locus gene which codes for the extracellular portion of a class I histocompatibility antigen.     

Cyclin synthesis drives the early embryonic cell cycle

We have produced extracts of frog eggs that can perform multiple cell cycles in vitro. Destruction of the endogenous messenger RNA arrests the extracts in interphase. The addition of exogenous cyclin mRNA is sufficient to produce multiple cell cycles. The newly synthesized cyclin protein accumulates during each interphase and is degraded at the end of each mitosis.     

Creative blocks: cell-cycle checkpoints and feedback controls.

Before division, cells must ensure that they finish DNA replication, DNA repair and chromosome segregation. They do so by using feedback controls which can detect the failure to complete replication, repair or spindle assembly to arrest the progress of the cell cycle at one of three checkpoints. Failures in feedback controls can contribute to the generation of cancer.     

Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor

Angiotensin II elicits different responses which affect cardiovascular, neuronal and electrolyte transport regulation. To understand the mechanisms responsible for its various actions, the receptor for angiotensin II has long been sought, but numerous attempts to purify the receptor have been unsuccessful owing to its instability and low concentration. We report here the expression cloning of a complementary DNA encoding a bovine angiotensin II receptor to overcome these difficulties. The receptor cDNA encodes a protein of 359 amino-acid residues with a transmembrane topology similar to that of other G protein-coupled receptors. COS-7 cells transfected with the cDNA expressed specific and high-affinity binding sites for angiotensin II, angiotensin II antagonist and a non-peptide specific antagonist for type-1 receptor. Dithiothreitol inhibited ligand binding. The concentration of intracellular Ca2+ and of inositol-1,4,5-trisphosphate increased in the transfected COS-7 cells in response to angiotensin II or angiotensin III, indicating that this receptor is the type-1 receptor for angiotensin II. Northern blot analysis revealed that the messenger RNA for this receptor is expressed in bovine adrenal medulla, cortex and kidney.     

Botanical content of black-tailed jackrabbit diets on semidesert rangeland

Botanical content of black-tailed jackrabbit diets was determined by microhistological examination of fecal samples collected from six different vegetation types in southern New Mexico on three dates. Grasses comprised the largest component of the jackrabbit diets, with dropseed species ( Sporobolus spp.) and black grama ( Bouteloua eriopoda ) the most abundant grasses in the diets. Leatherweed croton ( Croton pottsii ) and silverleaf nightshade ( Solanum elaeagnifolium ) were important forbs on most vegetation types. Diet composition varied in response to season and vegetation type. Grasses were important during the summer growing season, while forbs were selected during their growing season (summer or winter-spring). Shrubs were less abundant in the diet than grasses and forbs.