Medida del ángulo de incidencia de la irradiación solar directa

All five known human FMO genes have been localized to the long arm of chromosome 1 (Iq) (84,104,171,172). In each case, this was achieved by means of screening panels of human-rodent somatic cell hybrids by the polymerase chain reaction (PCR) technique. It is worth briefly considering the nature of somatic cell hybrids, given their importance to the experimental work described later in this thesis. Somatic cells in culture will only fuse infrequently to form a binucleate heterokaryon. It was discovered, however, that certain viruses with similar lipoprotein envelopes to the plasma membranes of human cells, such as the Sendai virus, have properties that can accelerate this process. Unlike most viruses, the Sendai virus makes numerous points of contact with the membrane of its host cell via a glycoprotein in the viral membrane. If two cells are positioned close to each other then the virus can simultaneously attach to both of them. As the virus is so small in comparison to both cells, they are held very close to each other. In many cases this will result in the membranes of the two cells fusing, producing a single binucleate heterokaryon. If, for example, a

suspension of human fibroblasts are mixed with mouse tumour cells in the presence of Sendai virus (inactivated by ultraviolet light), then the virus mediates fusion of both cell types. Incidentally, it has been found that the addition of polyethylene glycol, which causes cell plasma membranes to adhere to those of adjacent cells, produces the same effect without the need for a virus. Following cell fusion, both nuclei eventually fuse to form a uninucleate cell line. Through a process that is poorly understood, as the cells divide, human chromosomes are gradually eliminated in a random manner. A way of arresting this process, before all human chromosomes are lost, is to use mouse cells genetically deficient in some function (often a nutritional one) so that, if continued growth is to occur, the function must be supplied by the human genome. Such a selective technique allows the maintenance of hybrid cells that have a complete set of mouse chromosomes and a small number, or even just one, human chromosome(s). Sometimes, a human chromosome may not be complete and consist only of the long or short arm. The development of stains such as quinacrine and Giemsa, that generate a highly specific and constant pattern of banding for each chromosome at metaphase, allows rapid identification of the remaining human chromosome(s). Once the karyotype has been established, then the cell line can be maintained. Eventually, a battery of cell lines, each with a different complement of human chromosome(s), become available for analysis. If the presence of a human gene of interest is screened for from a panel of these cell lines, either through seeking to identify the presence of the gene product (for example, by testing a cell line biochemically for a particular enzyme or immunologically with a specific antibody preparation), or the gene itself (via hybridization techniques or PCR), then by a process of logical deduction the human chromosome (or chromosome arm) on which the gene resides can be determined. Human-rodent (rat, mice and hamster) somatic cell hybrids have been extensively used in this manner for somatic-cell genetic research. The development of somatic cell hybrids has revolutionized the mapping of human chromosomes ( 173- 177) . Prior to their development, geneticists had to rely on the study of family pedigrees in order to deduce linkage of various traits.

Shephard et a l refined the localization of human FMOl further to lq23-25 by

in situ hybridization of human FMOl cDNA to human metaphase chromosome

spreads. In this thesis, I will show how yeast artificial chromosomes, bearing human genomic DNA inserts that were known to contain human FMO genes, were used to further localize these genes by fluorescence in-situ hybridization (FISH) analysis of human metaphase chromosomes. The results obtained show that human FM O l,FM 02,FM 03 and FM 04 are clustered together at lq23-24,

whereas FM 05 is further removed towards the centromere at lq21. It is intriguing to speculate on the possible significance of these findings, such as whether the clustering of FMOl FM02, FM03 and FM 04 has any bearing on the regulation of their expression, or if it is mere coincidence that FM05 is both genomically separated from other members of the FMO gene family and unique in its apparent status as a non-drug metabolizing enzyme (140, 141). Certainly, it would seem unlikely that these genes would remain so tightly linked since their divergence from the common ancestral gene were it not for the fact it was an intrinsic requirement for their normal function. If one looks at the cytochromes P450 gene family, for example, members of this family that apparently diverged more recently than the FMOs are located on different chromosomes (178-180). It is tempting to draw comparisons with other more characterized gene families, such as the p-like globin gene cluster: in this instance, five functional genes (in addition to two non functional pseudogenes) are located within a region of about 50kb on human chromosome 11 and members of this family are expressed in a tissue- and developmental-specific manner (181). Also, from the standpoint of understanding the evolution of the FMO gene family, it would be interesting to elucidate the genomic locations of FMO genes in other mammalian organisms. To date, there appears to have been no reports of this, however.