Medio ambiente cultural o conductual:
12 TABLA 3 Características de la cohorte de mujeres con LES que
A wide variety of physical mapping strategies have been used to analyse the DNA of complex eukaryotic genomes. These can be dassed into low resolution physical mapping, where the smallest map unit that can be resolved is typically one to several megabases of DNA and high resolution physical mapping, where the resolution is typically very high, from hundred of kilobases to a single nudeotide. The advent of somatic cell hybridisation gave a major impetus to human gene mapping and the more recent molecular techniques using restriction enzymes, doned DNA probes, and high- resolution in situ hybridisation have greatly accelerated the process.
1.4.1 Low Resolution Physical Mapping
1.4.1.1 Somatic cell hybrids
Somatic cell hybrid panels can permit chromosomal localisation of any human DNA sequence. Under certain experimental conditions, cultured cells from different species can be induced to fuse together, thereby generating somatic cell hybrids. In human genetic mapping, hybrid cells are typically constructed by fusing human cells and rodent (usually mouse or hamster) cells. The initial fusion products described as heterokaryons, proceed to mitosis, resulting in dissolution of the two nuclear envelopes. The human and rodent nucleus are then brought together in a single nucleus. The hybrid cells are unstable initially; for reasons that remain unknown, most human chromosomes fail to replicate in subsequent rounds of cell division and are lost. This gives rise eventually to a variety of more or less stable hybrid cell lines, each with a full set of rodent chromosomes plus a few human chromosomes. The loss of the human chromsomes occurs essentially at random but can be controlled by selection.
The human chromosomes in somatic cell hybrids can be identified by PCR screening with sets of chromosome-specific primers (Abbott and Povey, 1991). By collecting hybrid cell lines with different sets of human chromosomes, it is possible to generate a hybrid cell panel that can be used to map any human DNA sequence to a specific chromosome. Localisation of a human DNA sequence to a single chromosome can be inferred by deduction: the chromosome must be common to all cell lines which are positive for the test and absent hrom all cell lines which are negative for the test, that is all hybrids should be concordant (Faraco et al, 1995). A disadvantage of traditional somatic cell hybrids is that the hybrid cells generally contain several human chromosomes rather than just a single human chromosome. In order to limit the amount of human genetic material transferred to a recipient rodent cell, the technique of microcell fusion (Fournier and Ruddle, 1977) can be applied. Microcell hybrids produced in this manner contain a few donor chromosomes, but the simplest contain a single donor chromosme (monochromosomal hybrids) (Warburton et al., 1990). Chromosomal localisation of human DNA clones can be established directly and rapidly using such monochromosomal hybrid panels which provide unambiguous evidence for the presence or absence of a given marker on a specified chromosome.
In order to obtain sub-chromosomal localisations, specialised hybrids are required which contain only part of a given human chromosome. The sub-chromosomal fragments may result from spontaneous chromosome breakage as a result of translocations or deletions, or they may be artificially induced. Cell lines are made from hybrids which contain the abnormal chromosome but which lack the normal homologue
of the chromosome of interest (translocation hybrids and deletion hybrids) (Abrams
et al., 1995). For this approach to be useful for a given chromosome, several different natural breakpoints must be available for that chromosome, a condition which may not always be met. One technique which enables genome-wide mapping involves the artificial breaking of human chromosomes and transfer of the sub-chromosomal fragments into rodent cells. Chromosome-mediated gene transfer (CMGT), is a procedure in which fragments of purified chromosomes are transferred into recipient cells in the presence of calcium phosphate (Porteous, 1987). Hybrids established by this method retain sub-chromosomal segments of human DNA (transgenomes) of a size that is useful for mapping (usually in the range of 1-50 Mb).
Irradiation fusion gene transfer is another method by which chromosome fragments are generated artificially, in this case by lethal irradiation of donor cells which are rescued by fusion with suitable recipient cells (Walter and Goodfellow, 1993). This method has been popular for use on somatic cell hybrids containing a single human chromosome. When DNA samples from a panel of such radiation hybrids are screened by hybridisation against a series of DNA clones, or by corresponding PCR assays, the patterns of cross-reactivity can be interpreted statistically to produce a linear map order for the DNA clones (Cox et al, 1990). A variant form of radiation hybrid mapping involves the use of human fibroblast cells as the starting donor cells instead of a monochromosomal somatic cell hybrid. This has the attraction of enabling construction of reasonably high resolution maps of the entire genome with a single panel of 100-200 hybrids (Walter et al, 1994).
1.4.1.2 In situ hybridisation
This is the most direct method of mapping. A cloned DNA fragment is hybridised directly to a spread of metaphase chromosomes, and the map location worked out by examining the result under the microscope (Gerhard et al, 1981). The sensitivity and resolution of in situ hybridisation has been increased significantly by the development of fluorescence in situ hybridisation (FISH) (Trask, 1991; van Ommen et al.,
1995). In this technique, a DNA probe is labelled by incorporation of modified nucleotides, obtained by covalent binding of a reporter molecule such as biotin or digoxigenin, which can be detected by specific binding to another molecule such as modified nucleotides. FISH has the advantage of providing rapid results which can be scored conveniently by eye using a fluorescence microscope.
A special application of FISH has been the use of DNA probes where the starting DNA is composed of a large collection of different DNA fragments fi*om a single type of chromosome. Such probes can be prepared by combining all human DNA inserts in a chromosome-specific DNA library (Davies et al., 1981). The resulting hybridisation signal
represents the combined contributions of many loci spanning a whole chromosome and causes the whole chromosome to fluoresce (chromosome painting). A few diflerently coloured fluorescence labels (chromosome paints) can be used in different ratios to provide numerous different colours for labelling chromosomes, thereby providing a molecular karyotype (Dauwerse et ah, 1992). The technique of chromosome painting has been extended recently by the ability to paint sub-chromosomal regions, using a mixed DNA probe corresponding to a particular sub-chromosomal region, as obtained from chromosome microdissection DNA libraries (Ludecke et al, 1989). Chromosome painting has found increasing applications in defining de novo rearrangements and marker chromosomes in clinical and cancer cytogenetics.
1.4.2 High Resolution Physical Mapping
1.4.2.1 Pulsed h eld gel electrophoresis (PFGE)
Restriction mapping permits molecular mapping with resolutions which depend on the frequency of the enzyme recognition site. Most restriction enzymes which recognise a 4- or 6-bp sequence typically cut vertebrate DNA once every few hundred or few thousand base pairs. The recognition sequences for rare-cutter restriction nucleases are typically 6-8 bp long and contain one or more CpG dinucleotides which are rare in vertebrate DNA (Bird, 1986). As a result, they generate fragments that are typically several kilobases in length. Whilst small restriction fragments can be size fractionated conveniently by agarose gel electrophoresis, large DNA fragments will remain unresolved. In order to separate such large restriction fragments, pulsed-field gel electrophoresis (PFGE) (Schwartz and Cantor, 1984), a modified form of agarose gel electrophoresis, is used. Large DNA fragments are separated in PFGE because the DNA molecules are subjected alternately to two approximately perpendicular fields. The presence of a discontinuous electric field means that the DNA molecules are intermittently forced to change their conformation and direction of migration during their passage through the gel. The time taken for a DNA molecule to alter its conformation and re-orientate itself in the direction of the new electric field is strictly size dependent. As a result DNA fragments up to several megabases in size can be fractionated efficiently. The resolution depends on the time of switching between the two fields (pulse time). Longer pulse times will resolve DNA fragments in the higher molecular weight range. Modifications of the original PFGE method have been developed which result in the DNA running straight in the tracks thus improving the resolution. The field is generated by the use of contour-clamped electric fields and the apparatus consists of an hexagonal array of electrodes to create a more homogeneous
field (Chu et al., 1986). The resolving power can then be optimised for fragments between 1 and 10 Mb.
A long-range restriction map of a chromosomal region can be generated by the same principle as conventional restriction mapping (Barlow and Lehrach, 1987). Physical linkage between two markers can be established if at least two restriction fragments are identical in size and a detailed map can be built up from information on single and double digests. Partial digests which are able to generate maps over much larger regions and to link more widely-spaced DNA probe have also been used in PFGE analyses. PFGE can also be used to identify candidate genes and assist in their isolation if the disease is caused by DNA deletion in some patients. The detection of deletions by PFGE has been useful in the diagnosis of carriers in Duchenne Muscular Dystrophy (DMD) (van Ommen et al., 1987).
1.4.2.2 Assem bly of clone contigs
In order to provide a framework for the ultimate physical map a series of cloned DNA fragments need to be assembled which collectively provide full representation of the sequence of interest. For a complete representation, with no gaps, the series of clones should contain overlapping inserts forming a comprehensive clone contig. In principle, contig assembly is facilitated by the way in which genomic DNA libraries are constructed: as part of the strategy for maximising the representation of a library, the genomic DNA is deliberately subjected to partial digestion with a restriction endonuclease. As a result, individual genomic DNA clones usually contain DNA sequences that partially overlap with those found in at least some other clones in the library. In order to identify clones with overlapping inserts, a specific DNA probe from one clone is used to screen a DNA library. With genomic DNA libraries, this permits the assembly of a clone contig by bidirectional chromosome walking from a fixed starting point.
Using arrayed libraries of YAC clones representing multiple copies of the genome, individual clones are isolated either by screening gridded arrays by hybridisation with single-copy probes (Nizetic et al., 1991; Bentley et al., 1992), or by testing pools of clones in multiple tiers or multiple arrays for the presence or absence of a sequence tagged site (STS) using a PCR assay. In early studies using these approaches, Y AGs were isolated and overlapped on the basis of their shared STS or probe content, and contiguous regions of several megabases were obtained (Anand et ah, 1991; Monaco
et ah, 1992). Where landmarks did not detect overlaps between clones directly, methods to isolate new landmarks from the ends of YAC were developed to facilitate chromosome walking in YACs. Frequently used methods include inverse-PCR (Ochman
techniques have been used to type clones at random and then integrate the information in order to identify clone contigs over large regions of a genome (Bellanne-Chantelot
et al, 1992). This approach has been used to great effect in assembling YAC contigs over significant amounts of the human genome (Cohen et al., 1993). Sequence-tagged site content mapping has also been used effectively to identify YACs with overlapping sequences (Cole et al, 1992; section 1.2.2).