Genes for the major protein antigens of the leprosy parasite Mycobacterium leprae

Leprosy, a chronic infectious disease afflicting between 10 and 15 million people, is caused by the obligate intracellular parasite Mycobacterium leprae. Although M. leprae was the first identified bacterial pathogen of man, basic biochemical, immunological, diagnostic and therapeutic investigations have been severely limited because it remains one of the few human pathogens that have not been cultured in vitro. An M. leprae recombinant DNA expression library was constructed to provide a source of genes encoding proteins relevant for such studies. Monoclonal antibodies directed against M. leprae specific antigens have been used to isolate the genes encoding the five most immunogenic protein antigens of the leprosy bacillus. We report here that M. leprae specific epitopes recognized by all of 13 monoclonal antibodies tested were produced by recombinant phage in Escherichia coli.     

A radiation hybrid map of the rat genome containing 5,255 markers.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.     

Energetic landscape of alpha-lytic protease optimizes longevity through kinetic stability.

During the evolution of proteins the pressure to optimize biological activity is moderated by a need for efficient folding. For most proteins, this is accomplished through spontaneous folding to a thermodynamically stable and active native state. However, in the extracellular bacterial alpha-lytic protease (alphaLP) these two processes have become decoupled. The native state of alphaLP is thermodynamically unstable, and when denatured, requires millennia (t1/2 approximately 1,800 years) to refold. Folding is made possible by an attached folding catalyst, the pro-region, which is degraded on completion of folding, leaving alphaLP trapped in its native state by a large kinetic unfolding barrier (t1/2 approximately 1.2 years). alphaLP faces two very different folding landscapes: one in the presence of the pro-region controlling folding, and one in its absence restricting unfolding. Here we demonstrate that this separation of folding and unfolding pathways has removed constraints placed on the folding of thermodynamically stable proteins, and allowed the evolution of a native state having markedly reduced dynamic fluctuations. This, in turn, has led to a significant extension of the functional lifetime of alphaLP by the optimal suppression of proteolytic sensitivity.     

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.     

The Mathematics of the Area Law: Kepler's Successful Proof in Epitome Astronomiae Copernicanae (1621)

Epitome V (1621), and consisted of matching an element of area to an element of time, where each was mathematically determined. His treatment of the area depended solely on the geometry of Euclid's Elements, involving only straight-line and circle propositions – so we have to account for his deliberate avoidance of the sophisticated conic-geometry associated with Apollonius. We show also how his proof could have been made watertight according to modern standards, using methods that lay entirely within his power. The greatest innovation, however, occurred in Kepler's fresh formulation of the measure of time. We trace this concept in relation to early astronomy and conclude that Kepler's treatment unexpectedly entailed the assumption that time varied nonuniformly; meanwhile, a geometrical measure provided the independent variable. Even more surprisingly, this approach turns out to be entirely sound when assessed in present-day terms. Kepler himself attributed the cause of the motion of a single planet around the Sun to a set of `physical' suppositions which represented his religious as well as his Copernican convictions; and we have pared to a minimum – down to four – the number he actually required to achieve this. In the Appendix we use modern mathematics to emphasize the simplicity, both geometrical and kinematical, that objectively characterizes the Sun-focused ellipse as an orbit. Meanwhile we highlight the subjective simplicity of Kepler's own techniques (most of them extremely traditional, some newly created). These two approaches complement each other to account for his success. (Received April 19, 2002) Published online April 2, 2003 Communicated by N. M. Swerdlow