Genetics of childhood disorders: VII. Cloning genes of interest.

An estimated 100,000 genes lie on our 46 chromosomes. One third of these are thought to be specifically expressed in our brains at different periods over the course of our lives. Over the past few years, a small but growing number of genes have been cloned. Mutations in a number of these genes lead to a disruption of the normal CNS developm ent . The ability to isolate genes is probably one of the most important recent achievements in neuroscience. This is a necessary first step before investigators are able to und erstand the normal role of these genes and how their protein products function in our brains. In the next several columns, we will discuss exactly how genes are cloned and how researchers learn more about the structure and function of the prot eins they encode. Last month, we reviewed how cytogenetic techniques have advanced to the point at which we can actually visual ize chrom osomal abnormalities such as a deletion.These physical disrupt ions point to the site on the chromosome that presumably has a mutation that contributes to the observed phenotype in the affected individual. These types of cytogenetic analyses reduce the search for a mutated gene from the several billion nucleotides on our total complement of DNA down to the millions of nucleotides contained within the deleted site. Considerable work remains, however, before the gene of interest is actually isolated, and various methods have been developed over the past decade to do exactly that. Mo st of the techniques to isola te genes make use of "libraries." Two types of libraries will be described. The first is called a complementary DNA (cDNA) library. This type of library is produced from-and therefore contai ns-all the messages expressed in a given tissue. To const ruct such a library, one first isolates the pool of RNA messages from a tissue, copies these messages into the more stable cDNA molecule, and places the cDNA molecules into an appropriate vector. Several types of vectors exist, but in general they serve similar functions: to carry a single cDNA molecule and to facilitate their own replication , thereby allowing the investigator to make many copiesof the library A single message, or "insert," is carried in each vector. Each vector and its insert is termed a clone, and it is the total collection of cDNA clones that is called the library. cDNA libraries consist of all the expressed sequences from a tissue of interest. The library will contain many copies of the most common housekeeping genes as these genes are abundantly expressed and many copies of their RNA messages were originally present when the RNA was isolated to make the library. T he library will contain fewer copies of less commonly expressed genes. However, if the screening methods are powerful , one should be able to find even very rarely expressed messages. The ultimate goal in screening a cDNA library is to isolate a clone of int erest. One then determines the nucleotide sequence for that clone, and the nucleotide sequence defines the precise amino acid sequence for the prote in the gene encodes. It should be noted that one can construct a cDNA from any tissue in the body. cDNAs can also be made from the same tissue at different time periods. For example, to identify genes that are transiently expressed dur ing the development of the cerebral cort ex, one would obtain cerebral cortices early in development, isolate the pool of RNA messages present in that tissue, convert these messages into cDNA molecules, and place the cDNA moleculesinto an appropriate vector.The end result would be a cerebral cortex cDNA library from early in development. Many of the clones contained in this library would also be found in an adult cerebralcortex cDNA library,as many genes are expressed at both times. However, the fetal cDNA library would also contain unique messages that are likely to be involved in the early development of the cerebralcortex. A second type of library is called a genom ic library. In this case, the goal of the investigator is to make a library from the total amount of chromosomal DNA. The DNA molecules are digested into smaller, more manageable pieces, and these smaller DNA sequ ences are placed into a vector. Special vectors have been constructed for this purpose, as the amount of DNA that must be packaged is considerably larger than for cDNA libraries. It is possible to replace large fragments of yeast chromosomes with segments of DNA from human chromosomes and have these segments replicated by the yeast. The library that is constructed using this type of vector is called a yeast artificial chromosome (YAC) library (Fig. 1). It allows researchers to package up to 1 million nucleotides into a vector. This larger stretch of genomi c DNA may contain several genes, each with their respective exons, int rons, and regulatory regions. It has been appreciated for some time that many housekeeping genes have particul ar repetitive sequences near their regulatory regions. These sequences are enriched for a partie-