In light of the key role of IL-5 in eosinophil development, understanding the mechanisms that regulate the activities of this
cytokine is critical to the development of strategies for therapeutic intervention in various clinical conditions associated with
eosinophilia. The regulation of the biological activities of IL-5 can be accomplished at four levels: 1) regulation of its secretion; 2) regulation of its receptor expression; 3) regulation of its activity by other cytokines, as seen in the reciprocal cross-inhibition of Thl/Th2 subsets; and 4) regulation by soluble inhibitors, either soluble receptors or antagonists.
The aim of the project described in this thesis was to characterise the mouse IL-5 receptor a-chain gene and to study the mechanisms regulating its expression in a cell type-specific manner. This involved the isolation and complete sequence determination of the IL-5Ra gene and analysis of its promoter region to identify sequences involved in cell type-specific regulation. In addition, constructs were also prepared for use in altering the endogenous IL-5 receptor a-chain gene in mice by homologous recombination. One construct was designed to
inactivate the endogenous gene and the other to generate mice that only express the transmembrane form of the receptor and not the soluble form. The aim here is to further study the function of both forms of the receptor in vivo.
CLONING AND SEQUENCING OF THE
MOUSE IL-5R0C GENE
2.1 Introduction
The development of vectors, such as bacteriophage X and PI,
cosmids and yeast artificial chromosomes (YAC), that have the capacity
to accomm odate large inserts has facilitated the construction of
representative genomic libraries for the mapping and analysis of complex genomes. The usual approach to isolating a gene is to screen a recombinant genomic DNA library consisting of several million individual clones. Each clone contains a different segment of the genome. A cloning strategy for isolating the clone containing the desired gene is formulated according to the information available for the particular gene and the strategy used for determining the sequence of the isolated gene is dependent on its size and complexity.
2.1.1 Screening strategy
A genomic library can be screened for the presence of a desired gene by hybridisation to a nucleic acid probe or by detection of specific gene fragments using the polymerase chain reaction (PCR) (reviewed by Maniatis et al 1989, Ausubel et al. 1994). The nucleic add probe used can either be a fragment of the gene, its cDNA or a gene-specific
oligonucleotide. Normally, a gene fragment or cDNA is preferred over an oligonucleotide because they can be labelled to much higher specific activity, thus, enhancing the sensitivity of detection. Gene detection by PCR is simple and more rapid because the results of a PCR screen are known on the same day without the need for lengthy autoradiography. However, the PCR procedure suffers from the drawback of not being able to detect overlapping dones if the size of the gene is greater than the capacity of the cloning vector. Nevertheless, it is useful for
detecting specific gene fragments and serves as a supplementary screening procedure to the use of nucleic acid probes.
2.1.2 Sequencing strategy
The amount of sequence data that can be obtained from a sequendng reaction is limited by the resolution of the sequencing gel. Current technologies allow about 500 nucleotides of sequence data to be reliably obtained from one set of sequendng reactions. The
characterisation of long regions of DNA necessitates the breaking of a large fragment into smaller ones. The manner in which these smaller fragments are generated is the fundamental issue in the choice of the
overall sequencing strategy. Current strategies fall into two general categories: random and directed.
The random or shotgun strategy generates a library of smaller fragments from a large fragment of DNA after random digestion with DNase I (Anderson 1981) or physical shearing by sonication (Deininger 1983; Bankier et al 1987). The individual smaller fragments are
sequenced randomly and ordered by finding overlaps. This approach has the advantage of rapid initial accumulation of data but may suffer at the end of the project from repeated sequencing of the same regions and from difficulties in closing remaining gaps. It has nevertheless been successfully used for large sequencing projects such as the
C. elegans genome project and gives data on both strands (Sulston et al.
1992).
The directed strategy allows direct and sequential sequence analysis of large DNA fragments from one end to another. The two basic approaches used in this strategy are primer walking and
progressive (nested) deletions. In primer walking, the initial sequence is determined by a vector-specific primer and then followed by
synthesis of a new primer from the newly acquired sequence. These steps are repeated until the DNA fragment is completely sequenced (Strauss et al. 1986). One nested deletion approach uses exonuclease
in
to generate progressive unidirectional deletions from one end of the DNA fragment (Henikoff 1984). The drawback of this method is the requirement that the DNA fragment be free of the restriction enzyme site used to linearize the vector prior to exonuclease HI treatment.2.1.3 Overview of this chapter
The aim of the work described in this chapter was to clone and sequence the mouse IL-5Ra gene. This was essential for the study of its regulation and for its manipulation by gene targeting. It was decided to determine the complete sequence of the gene rather than just the intron/exon junctions in order to provide a sound basis for a
comprehensive study of the gene and its regulation. In addition, knowledge of the complete sequence of the IL-5Ra gene would greatly facilitate manipulation of the gene in the construction of targeting vectors designed to generate mice in which the IL-5Ra gene had been modified by gene targeting.