A model system for studying the integration of molecular biology databases

MOTIVATION: Integration of molecular biology databases remains limited in practice despite its practical importance and considerable research effort. The complexity of the problem is such that an experimental approach is mandatory, yet this very complexity makes it hard to design definitive experiments. This dilemma is common in science, and one tried-and-true strategy is to work with model systems. We propose a model system for this problem, namely a database of genes integrating diverse data across organisms, and describe an experiment using this model. RESULTS: We attempted to construct a database of human and mouse genes integrating data from GenBank and the human and mouse genome-databases. We discovered numerous errors in these well-respected databases: approximately 15% of genes are apparently missing from the genome-databases; links between the sequence and genome-databases are missing for another 5-10% of the cases; about a third of likely homology links are missing between the genome-databases; 10-20% of entries classified as 'genes' are apparently misclassified. By using a model system, we were able to study the problems caused by anomalous data without having to face all the hard problems of database integration. CONTACT: nat@jax.org     

A Bayesian approach to validating STR multiplex databases for use in forensic casework

A previous paper in this journal has described the conventional statistical analysis of three databases (Caucasian, Afro-Caribbean and Asians from the Indian subcontinent) where individuals are typed at six short tandem repeat (STR) loci. This paper presents a Bayesian analysis of the same data and the approach is centred on the concept of estimating coancestry coefficients from mixed databases. Posterior distributions for the three databases are presented and the discussed and the consequences of implementing bootstrap estimation procedures are also shown.     

Introduction to the techniques of molecular biology

The purpose of this chapter is to outline some of the common recombinant DNA methods in use today. These techniques are usually employed to isolate a defined portion of the genome, mostly a gene, from an organism or tissue of interest and, thereafter, to characterize the structure and function of this genetic material. To isolate a gene, genomic DNA is extracted from a selected tissue. For a better handling the relatively large DNA molecules are cut into a mixture of fragments by restriction endonucleases. The fragments are then separated from each other according to their size by gel electrophoresis. A procedure called Southern blotting is used to verify the presence of the desired gene in one of the DNA fragments separated on an agarose gel. The DNA fragments are transferred from the gel to a filter whereby the original fragment pattern is maintained. Then, a single-stranded DNA or RNA probe specific for the gene to be isolated is hybridized to its target fragments fixed to the filter. A radioactive or fluorescent tag is attached to the probe for subsequent identification. In cases where only transcribed sequences are to be isolated cytoplasmic messenger RNA (mRNA) is prepared instead of DNA. Analysis of RNA by a technique similar to Southern blotting is termed Northern blotting. Preservation of DNA sequences is usually achieved by DNA cloning. DNA cloning involves the insertion of a DNA fragment into a DNA vector and the stable incorporation of the recombinant DNA into a suitable host. Propagation of the host facilitates the amplification of the recombinant DNA for subsequent analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carcinogenesis: from epidemiology to molecular biology

Carcinogenesis is a multi-step process and at least three stages can be distinguished: initiation, promotion and progression. Initiation is made up of events whereby an exogenous or endogenous carcinogen induces alterations in the genome of the cell; this results in a lesion that can be inherited and which confers upon that cell the potential for neoplastic growth. This is the result of activation of cellular proto-oncogenes into transforming oncogenes and/or the inactivation of the two alleles of tumor suppressor genes (anti-oncogenes). Although it is conceivable that a single event or a single lesion can initiate a cell it is probable that in most cases initiation is the consequence of a series of events since the different types of proto-oncogenes do not function independently of each other. The coordinated activation of more than one oncogene is generally required to confer the full potential for neoplastic growth. DNA repair and apoptosis play a critical role in carcinogenesis and the effectiveness of these mechanisms might be influenced by the dose and the time interval between individual events (dose rate). Epidemiological and experimental data show also the main role of cell proliferation after initiation. Proliferation influences the probability of DNA repair and contributes to further genomic alteration. Cell to cell interaction within a tissue participates in the control of cell proliferation and cell repair. The evolution of one initiated cell is not independent of the surrounding cells and disorganization of a tissue is a factor in carcinogenesis. Presently carcinogenesis appears to be a complex phenomenon which cannot be a adequately modelled. The linear no-threshold model has been used for the purpose of cancer prevention and legal norms, however it should be realized that its experimental and theoretical bases are debatable and that much recent data do not support them. Its predictions should therefore be considered with great caution. All efforts should be made in the future to build new models incorporating available epidemiological and fundamental data.     

A molecular biological approach to synaptic plasticity and learning

The antigen receptors on the surface of B- and T-lymphocytes are complexes of several integral membrane proteins, essential for their proper expression and function. Recent studies demonstrated that transmembrane (TM) domains of the components of these receptors play a critical role in their association and function. It was specifically demonstrated that in many cases point mutations in the TM domains can partially or completely disrupt the receptor surface expression and function. Here we review studies of the TM domains of B- and T-cell receptors. Furthermore, we use a novel method, PHDtopology, to provide estimates of the exact locations and lengths of the TM domains of the subunit components of these receptors. Most previous studies used single residue hydrophobicity as a criterion for determining the position and length of the TM domains. In contrast, PHDtopology utilizes a system of neural networks and the evolutionary information contained in multiple alignments of related sequences to predict the location, length, and orientation of transmembrane helices. Present results significantly differ from most published estimates of the TM domains of the B- and T-cell receptor components, primarily in the length of the TM domains. These results may lead to modification of putative TM motifs and re-interpretation of the results of studies using mutated TM domains. The availability of PHDtopology on the Internet would make it a valuable tool in the future studies of the TM domains of integral membrane proteins.     

New contributions to clinical hypertension from molecular biology

The authors constructed a new dynamic guiding splint assisting the active mobilisation after flexor tendon repair distal to the wrist. In these cases, the "inverse" wrist position seems to be the best position for mobilisation. This means that finger flexion should be carried out during wrist extension, and finger extension during wrist flexion. The splint guides and co-ordinates the movements of the wrist and the fingers, and it limits the free usage of the hand.     

A systematic molecular genetic approach to study mammalian germline development

It is difficult to study gene expression in mammalian embryonic germ cells as PGCs constitute only a minor proportion of the mouse embryo. We have overcome this problem by using a novel combination of established molecular and transgenic approaches. A line of mice has been generated in which the cells of the germ lineage express the beta-galactosidase reporter gene during embryogenesis. Using this line, germ cells have been purified to near homogeneity from embryos at discrete stages during germline development by use of a stain for beta-gal activity and a fluorescence activated cell sorter. Subsequently, cDNA libraries have been constructed from each germ cell population using a modified lone-linker PCR strategy. These combined cDNA libraries represent genes expressed in PGCs during mammalian germline development. To facilitate a molecular genetic approach to studying mammalian germline development, these cDNA libraries will be pooled to form an arrayed, addressed reference embryonic germ cell cDNA library. In parallel with large-scale cDNA sequencing efforts; genes that are differentially expressed in germ cells will be identified by screening the reference library with probes generated by subtractive hybridization. Complementary DNAs identified using this approach will be analyzed by sequencing, database comparison, genomic mapping and in situ hybridization to ascertain the potential functional importance of each gene to germline development. In addition to providing a wealth of novel information regarding patterns of gene expression during mammalian germline development, these results will form the basis for future experiments to determine the function of these genes in this process.     

A multivariate approach to affected-sib-pair analysis using highly dense molecular maps

Adaptation of the Bacillus subtilis strain 2335/105 (Km Inf+) containing a recombinant plasmid encoding the extracellular human interferon alpha 2 was studied under various conditions. Stability of the plasmid in the population of B. subtilis 2335/105 was estimated under nonselective conditions. The plasmid-free cells and cells with a low number of plasmid copies were found to accumulate progressively, constituting 80% of the population after 10 culture passages, indicating the poor competitiveness of cells carrying a high number of plasmid copies. The behavior of vegetative cells of the recombinant strain introduced into aquatic microcosms differing in trophic chain length was studied. Within the first 10 days, the lysis of vegetative cells of B. subtilis 2335/105 occurred; the number of viable spores was very low but remained constant for half a year.     

A universal approach to bacterial molecular epidemiology by polymerase chain reaction ribotyping

Oligonucleotide primers complementary to conserved regions of the 16S and 23S ribosomal RNA genes were used to amplify the 16S-23S intergenic spacer region of bacterial pathogens. The amplification patterns produced were compared for their potential use in molecular epidemiologic analysis. This method, polymerase chain reaction (PCR) ribotyping, was applied to isolates of Staphylococcus aureus, Enterococcus faecium, Escherichia coli, and Enterobacter species. Length polymorphisms in the amplified DNA distinguished unrelated strains of all bacteria. The banding patterns of 3 S. aureus isolates from the blood of 1 patient on 3 consecutive days were identical. Plasmid analysis, biotyping, and antibiograms were also obtained on the Enterobacter isolates. All three of these methods showed considerable variability after in vitro passage of bacteria, but PCR ribotypes remained stable. Results demonstrate the utility of the conserved primers for PCR ribotyping, a widely applicable method for the molecular epidemiology of genetically diverse bacteria.     

A comment on information theory and central dogma of molecular biology

The technique of competitive double-labelling [H. Kaplan, K.J. Stevenson & B.S. Hartley, (1971) Biochem. J. 124, 289-299; L.P. Visentin & H. Kaplan (1975) Biochemistry 14, 463-468] was used to determine the reactivity of some amino groups towards acetic anhydride in deoxy-and liganded haemoglobin. Only those amino groups known to form salt bridges in deoxy-but not in liganded haemoglobin (i.e. the alpha-amino group of valine-1 alpha and the xi-amino group of lysine-40 alpha and lysine-127 alpha [M. F. Perutz (1970) Nature (London) 228, 726-739]) and different reactivities in the two structures.     

Contribution of molecular biology to the diagnosis of monogenic hereditary nephropathies

Schematically, gene identification can be achieved by functional cloning, based on preexisting knowledge about the basic biochemical defect, positional cloning, initiated by the mapping of the responsible gene to its correct location on a chromosome, or by a combination of these two approaches called "candidate gene" approach. Genes of numerous monogenic hereditary renal disorders have been identified during the last few years by one of these approaches, particularly, the PKD1 and PKD2 genes involved in autosomal dominant polycystic kidney disease, as well as the genes encoding different type IV collagen alpha chains, responsible for Alport syndrome. This allows novel insights in the understanding of the pathogenesis of hereditary renal diseases and has opened new areas of genetic diagnosis.     

A physical approach to reduce nonspecific adhesion in molecular recognition atomic force microscopy

Atomic force microscopy is one of the few techniques that allow analysis of biological recognition processes at the single-molecule level. A major limitation of this approach is the nonspecific interaction between the force sensor and substrate. We have modeled the nonspecific interaction by looking at the interaction potential between a conical Si3N4 tip with a spherical end face and a mica surface in solution, using DLVO (Derjaguin, Landau, Verwey, Overbeek) theory and numerical calculations. Insertion of the tip-sample potential in a simulation of an approach-retract cycle of the cantilever gives the well-known force-distance curve. Simulating a force-distance curve at low salt concentration predicts a discrete hopping of the tip, caused by thermal fluctuations. This hopping behavior was observed experimentally and gave rise to a novel approach to making measurements in adhesion mode that essentially works in the repulsive regime. The distance between tip and sample will still be small enough to allow spacer-involved specific interactions, and the percentage of nonspecific interactions of the bare tip with the mica is minimized. We have validated this physical model by imaging intercellular adhesion molecule 1 (ICAM-1) antigen with a tip functionalized with anti-ICAM-1 antibody. The measurement demonstrated that a significant decrease in the number of nonspecific interactions was realized, and the topographical image quality and the specific bonding capability of the tip were not affected.     

A. Weismann's germ-plasma theory: a new methodological approach to the problems of general biology

A. Weismann's theory of germ plasm is of special importance in the history of theoretical biology. Its meaning was not confined by presenting of neoperformistic ideas on the new level of science. In fact it predicted reduction division, the continuing of germ plasm and the significance of chromosomes in heredity. For the first time it brought a new methodology to the experimental genetics and the idea of interdisciplinary synthesis.     

Context, cortex, and dopamine: A connectionist approach to behavior and biology in schizophrenia.

Connectionist models are used to explore the relationship between cognitive deficits and biological abnormalities in schizophrenia. Schizophrenic deficits in tasks that tap attention and language processing are reviewed, as are biological disturbances involving prefrontal cortex and the mesocortical dopamine system. Three computer models are then presented that simulate normal and schizophrenic performance in the Stroop task, the continuous performance test, and a lexical disambiguation task. They demonstrate that a disturbance in the internal representation of contextual information can provide a common explanation for schizophrenic deficits in several attention- and language-related tasks. The models also show that these behavioral deficits may arise from a disturbance in a model parameter (gain) corresponding to the neuromodulatory effects of dopamine, in a model component corresponding to the function of prefrontal cortex. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

An introduction to molecular biology: gene structure, expression, and mutation

This mini-review outlines DNA structure and the regulation of gene expression. It then discusses the nature of mutations, techniques employed to detect them, and the limitations of these techniques.