Computer simulation study of molecular recognition in model DNA microarrays

DNA microarrays have been widely adopted by the scientific community for a variety of applications. To improve the performance of microarrays there is a need for a fundamental understanding of the interplay between the various factors that affect microarray sensitivity and specificity. We use lattice Monte Carlo simulations to study the thermodynamics and kinetics of hybridization of single-stranded target genes in solution with complementary probe DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct and each segment represents a sequence of nucleotides ( approximately 11 nucleotides). Each probe segment interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how the probe length, temperature, or hybridization energy, and the stretch along the target that the probe segments complement, affect the extent of hybridization. For systems containing single probe and single target molecules, we observe that as the probe length increases, the probability of binding all probe segments to the target decreases, implying that the specificity decreases. We observe that probes 12-16 segments ( approximately 132-176 nucleotides) long gave the highest specificity and sensitivity. This agrees with the experimental results obtained by another research group, who found an optimal probe length of 150 nucleotides. As the hybridization energy increases, the longer probes are able to bind all their segments to the target, thus improving their specificity. The hybridization kinetics reveals that the segments at the ends of the probe are most likely to start the hybridization. The segments toward the center of the probe remain bound to the target for a longer time than the segments at the ends of the probe.     

Mechanical regulation of secondary chondrogenesis

The development of the skull is characterised by its dependence upon epigenetic influences. One of the most important of these is secondary chondrogenesis, which occurs following ossification within certain membrane bone periostea, as a result of biomechanical articulation. We have studied the genesis, character and function of the secondary chondrocytes of the quadratojugal of the chick between embryonic days 11 and 14. Analysis of gene expression revealed that secondary chondrocytes formed coincident with Sox9 upregulation from a precursor population expressing Cbfa1/Runx2: a reversal of the normal sequence. Such secondary chondrocytes rapidly acquired a phenotype that is a compound of prehypertrophic and hypertrophic chondrocytes, exited from the cell cycle and upregulated Ihh. Pulse and pulse/chase experiments with BrdU confirmed the germinal region as the highly proliferative source of the secondary chondrocytes, which formed by division of chondrocyte-committed precursors. By blocking Hh signalling in explant cultures we show that the enhanced proliferation of the germinal region surrounding the secondary chondrocytes derives from this Ihh source. Additionally, in vitro studies on membrane bone periosteal cells (nongerminal region) demonstrated that these cells can also respond to Ihh, and do so both by enhanced proliferation and precocious osteogenesis. Despite the pro-osteogenic effects of Ihh on periosteal cell differentiation, mechanical articulation of the quadratojugal/quadrate joint in explant culture revealed a negative role for articulation in the regulation of osteocalcin by germinal region descendants. Thus, the mechanical stimulus that is the spur to secondary chondrocyte formation appears able to override the osteogenic influence of Ihh on the periosteum, but does not interfere with the cell cycle-promoting component of Hh signalling.     

Analysis of host preference and geographical distribution of Anastrepha suspensa (Diptera: Tephritidae) using phylogenetic analyses of mitochondrial cytochrome oxidase I DNA sequence data

Anastrepha suspensa (Loew) is an economically important pest, restricted to the Greater Antilles and southern Florida. It infests a wide variety of hosts and is of quarantine importance in citrus, a multi-million dollar industry in Florida. The observed recent increase in citrus infested with A. suspensa in Florida has raised questions regarding host-specificity of certain populations and genetic diversity of the pest throughout its geographical distribution. Cytochrome oxidase I (COI) DNA sequence data was used to characterize the genetic diversity of A. suspensa from Florida and Caribbean populations reared from different host plants. Maximum likelihood and Bayesian phylogenetic methods were used to analyse COI data. Sequence variation among mitochondrial COI genes from 107 A. suspensa samples collected throughout Florida and the Caribbean ranged between 0 and 10% and placed all A. suspensa as a monophyletic group that united all A. suspensa in a clade sister to a Central American group of the A. fraterculus paraphyletic species complex. The most likely tree of the COI locus indicated that COI sequence variation was too low to provide resolution at the subspecies level, therefore monophyletic groups based on host-plant use, geography (Florida, Jamaica, Cayman Islands, Puerto Rico or Dominican Republic) or population sampled are not supported. This result indicates that either no population segregation has occurred based on these biological or geographical distinctions and that this is a generalist, polyphagous invasive genotype. Alternatively, if populations are distinct, the segregation event was more recent than can be distinguished based on COI sequence variation.     

Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.     

Modularity and sense organs in the blind cavefish, Astyanax mexicanus

SUMMARY Mexican tetra (Astyanax mexicanus) exist as two morphs: a sighted (surface) form and a blind (cavefish) form. In the cavefish, some modules are lost, such as the eye and pigment modules, whereas others are expanded, such as the taste bud and cranial neuromast modules. We suggest that modularity can be viewed as being nested in a manner similar to Baupläne so that modules express unique sets of genes, cells, and processes. In terms of evolution, we conclude that natural selection can act on any of these hierarchical levels within modules or on all the sensory modules as a whole. We discuss interactions within and between modules with reference to the blind cavefish from both genetic and developmental perspectives. The cavefish represents an illuminating example of module interaction, uncoupling of modules, and module expansion.     

Cryptochromes: sensory reception, transduction, and clock functions subserving circadian systems

Cryptochromes (CRYs) are blue-light-absorbing proteins involved in a variety of biological phenomena. In animals, CRYs exhibit a certain versatility with regard to these organisms' circadian rhythms, as has been revealed by the effects of mutations and molecular manipulations. The rhythm system of Drosophila uses one gene's worth of CRY protein to transmit light into a circadian clock within the brain, which controls the fly's sleep-wake cycles. In fact, the relevant pacemaking neurons are themselves circadian photoreceptive structures. In peripheral tissues and others located posterior to the brain, Drosophila CRY may be a photoreceptive molecule and also part of the pacemaker mechanism. Mice have two CRY-encoding genes. They are expressed in many tissues, including the retina and a clock structure within the brain. In the former location, mouse CRY may play a circadian-photoreceptive role, along with that mediated by rhodopsins found elsewhere in the retina. In the latter tissue, the hypothalamic suprachiasmatic nucleus, mouse CRYs are closely connected to the multimolecule murine clock mechanism.     

The endocytic apparatus of the kinetoplastida. Part II: machinery and components of the system

Endocytic systems within eukaryotic cells are a diverse set of intracellular transport pathways responsible for uptake, recycling, interaction with the exocytic system and degradation of molecules. Each of these pathways requires the interaction of distinct protein components that function in macromolecule sorting, control of transport rates and in membrane biogenesis. In the second of two articles on kinetoplastida endocytosis, the endocytic system in Trypanosoma brucei is considered as a model, and the molecules that control this system and the protein components of the endocytic pathway are discussed. We also consider novel mechanisms for sorting that have been proposed to operate in trypanosomes.