Chromosome mapping in humans (including RFLPs, etc.)

During the last two decades, human species has become a very favourable material for genetic studies. This was despite the fact that in this material conventional genetic analysis was not possible, firstly, due to relatively small human family size, and secondly due to lack of possibility to make experimental matings. These difficulties in genetic analysis and chromosome mapping in human beings have largely been overcome by the development of methods for parasexual analysis based on fusion of cells in culture. It is thus obvious that indirect methods have been used for chromosome mapping in humans. Criteria used for this purpose include the following: (i) family linkage studies, (ii) segregation from cell hybrids, (iii) correlation of loss of marker from cell hybrids with radiation induced loss of chromosome segment, (iv) linkage disequilibrium in population with another mapped marker and (v) in situ hybridization. It may be noted that the basic technique, which has largely been used is the segregation in cell hybrids. Other techniques, mainly used correlation with already mapped genes or in situ hybridization, where mRNA, tRNA, or rRNA could be used for in situ hybridization on intact chromosomes, to find out the location of their genes.

Segregation in somatic cell hybrids Cell fusion. The first step in the study of segregation of somatic cell hybrids involving human cells, is the cell fusion. Induction of fusion of dissimilar cells involved the use of inactivated virus (Sendai virus). The presence of this virus even when killed induces cell fusion. If one has a technique to screen the fused hybrid cells, these fused cells can be obtained from mixed cell cultures even without virus treatment. These fused cells will be heterokaryons having all chromosomes from each of the participating cells. Such fused cells have unstable karyotypes and can lose whole chromosomes or chromosome segments without causing any damage to the function of the cell. This is due to presence of four fold redundancy (each chromosome will be in tetrasomic condition). Such lines could also be prepared where the loss of human chromosomes from man-mouse hybrids was preferential and non-random. In these lines, the loss of single specific human chromosome, could be studied by karyotype, so that by relating it with the loss of specific function, a gene can be located on the missing chromosome or its specific segment.

Production and selection of markers. Auxotrophs available in fungi, are difficult to obtain in human cell cultures. Moreover, the recessive mutations will not express themselves due to heterozygosity. The latter difficulty is overcome, since loss of chromosome with dominant allele in cell cultures may allow the recessive mutation to express. The mutations which affect the utilization of nucleosides or free bases for nucleotide synthesis have been used for this purpose. The free bases or nucleosides may be produced not as intermediate products in nucleotide synthesis, but are produced due to nucleic acid degradation. For cell economy they are used again with the help of a number of salvage enzymes. Some of these salvage enzymes include the following (i) HGPRT (hypoxanthine-guanine phosphoribosyl transferase) converts guanine into guanylic acid and hypoxanthine (inosine) into inosinic acid, (ii) APRT (adenine phosphoribosyl transferase) converts adenine into adenylic acid, (iii) TK (thymidine kinase), converts thymidine into thymidylic acid. A medium could be prepared, on which these three enzymes are essential for growth of cell hybrids. This was called HAT medium. If now a base analogue like bromodeoxyuridine (which was highly toxic if incorporated in the nucleic acid), was used in the medium, cells deficient for a salvage enzyme (thymidine kinase in this case) will prohibit incorporation and therefore, such deficient cells only will survive. These will be characterized as resistant to this toxic drug and will grow on HAT medium supplemented with thymidylic acid, since thymidine kinase is absent.

Assignment of markers to chromosomes.
If cells (mouse cell line) resistant to a toxic drug like bromodeoxyuridine are hybridized with normal human cells, the derived hybrid cells on repeated divisions in HAT medium lose all other human chromosomes except the one responsible for synthesis of thymidine kinase. The growth of cells on HAT medium takes place only when these cells synthesize thymidine kinase, besides other enzymes. This exceptional human chromosome was always found to be the same i.e. chromosome 17 and could be identified by banding technique. The gene for thymidine kinase could thus be located on chromosome 17 of humans.

Using resistant lines for several markers,independence or linkage between several genes could be worked out. Differences between human and mouse enzymes could also be worked out and a number of such enzymes could be identified. In these cases absence or presence of enzyme was not used, but instead migration of enzyme on electrophoretic gel, was used, as a criterion. Of the 17 enzymes, 14 could be lost independently of not only each other, but also independent of the remaining three enzymes which were always lost together. The genes for 14 enzymes thus should be located on separate chromosomes and the three linked genes for enzymes HGPRT, G6PD (glucose 6-phosphate dehydrogenase) and PGK (phosphoglycerate kinase) were later found to be present on X-chromosome.

By studying cell lines, which have lost only a part of a chromosome rather than the whole chromosome, genes could also be located on a specific region of a chromosome. The progress made in chromosome mapping in humans upto the year 1991 is summarized in Table 24.4. The progress in mapping of the longest human chromosome called chromosome-1 is shown in Figure 24.6. It is hoped that the human gene map should be completely defined by the end of present century, if not at the chromosomal level, then at the genetic level.


* Figures in parentheses are the numbers of disease related genes mapped
** Figures in parentheses are number of PCR based polymorphic markers (PCR = polymerase chain reaction; consult Genetic Engineering and Biotechnology 1.  Recombinant DNA and PCR (Cloning and Amplification of DNA)).

Genetic linkage map of human X-chromosome.
The linkage map of X-chromosome was prepared by first locating DNA markers using either a physical method due to in situ hybridization, or biochemical meth. j where marker DNA was hybridized on the DNA of individual hybrid cell lines, each containing a different portion of human X-chromosome. In the later case, absence of hybridization was used to infer that the marker is located on the missing segment of X-chromosome.

Gene distances and linear order of genes were determined by analysing families with the help of large number of DNA markers. The basis for using families was as follows. The X-chromosome from a maternal grandfather, without undergoing any recombination passes to the mother, where it undergoes recombination to be identified in children (boys and girls). Recombinant and non-recombinant chromosomes can be scored in children by DNA markers. From the data, gene distances and linear order of genes can be established. In a study conducted recently, genetic linkage relationships in X-chromosome were established among 21 DNA markers by examining DNA from 38 normal families each involving maternal and paternal grandparents, parents and children (an average of 9 children). As a result of this study, a linkage map of human X-chromosome was prepared which is reproduced in Figure 24.7.

RFLPs (restriction fragment length polymorphisms) as molecular markers.
In recent years RFLPs have been used as molecular genetic markers in human beings (see Genetic Engineering and Biotechnology 2.  Restriction Maps and Molecular Genetic Maps). These RFLPs were revealed using a number of restriction endonucleases for digestion of genomic DNA, followed by hybridization with specific genomic or cDNA clones (see Genetic Engineering and Biotechnology 1.  Recombinant DNA and PCR (Cloning and Amplification of DNA)) used as probes. The technique used for RFLP mapping in humans differs from those in mice, fruitfly or plants. Segregation patterns of the markers is studied within a panel of reference families (cell lines from 59 such families as a source of DNA have been maintained in Paris) and recombination frequencies calculated from this finite and limited sample size. Once RFLPs are ordered, models are fitted by maximizing the likelihood of parameters. The likelihoods of different models are compared by comparing likelihood ratios (1000 : 1, 100 : 1 or 10 : 1). The log₁₀ of this ratio is called LOD score (for 1000 : 1, LOD score = 3, for 100 : 1, LOD score = 2), which is used for linkage analysis. A LOD score equal to or exceeding 3 is a proof of linkage. Of 7000-8000 loci needed for a saturated map with a resolution of 1 cM, about 3000 loci have already been mapped (Table 24.4; for more details consult an advanced book on RFLPs or a recent book on human genetics).

Proposal for sequencing of human genome.
Earlier during the years 1986 and 87, there has been a debate among the molecular biologists about the desirability and feasibility of sequencing the nucleotides in the entire human genome. This will involve sequencing of 3 billion nucleotides and will need 30,000 person-years (expenses more than two billion U.S. dollars). The idea later gained momentum and the merits of sucha project were recognized. A part of the jenome has already been sequenced and the next few years will see the sequencing of the whole human genome (Table 24.5).