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Gene library

A term used to describe a collection of DNA fragments derived from the genome of an organism and cloned randomly into suitable cloning vectors (plasmids, phages). If plasmid cloning vectors are used for the establishment of the library, this library essentially is a collection of host cells, each of which contains a plasmid with an inserted DNA fragment. If phages have been used to clone the DNA fragments, the library consists of a phage lysate with each phage containing an inserted fragment of DNA. Together the individual fragments in the collection of inserted molecules represent the entire genetic information contained in an organism and in this case the library is said to be a representative library.

If the library has been established by using fragmented cellular DNA of an organism the library is said to be a genomic library. The term genomic DNA clone or chromosomal DNA clone then refers to an individual cell carrying a cloning vector with one of the cellular DNA fragments or to a phage isolate with a specific DNA insert. Subgenomic libraries are obtained if only selected portions of the genome, for example fragments from distinct sorted chromosomes, are represented in the library. A characteristic of genomic libraries is the fact that the individual inserts still contain non-coding intron sequences. Such libraries are used, therefore, for determining the genomic structures of genes.

Principle of gene cloning and preparation of gene libraries. A suitable cloning vector (in this example a circular plasmid) is linearized by cleavage with a suitable restriction enzyme (1). The cellular DNA containing the gene of interest (red) is also cleaved into fragments with this enzyme (2). For various reasons the cellular DNA is cleaved in a way yielding N overlapping fragments with a uniform length of 20 kb. Linearized vector DNA and genomic DNA fragments are then ligated in vitro, yielding a population of N recircularized cloning vector molecules, each of which contains an insert of cellular genomic DNA (3). These molecules are introduced into suitable host cells (4). The collection of cloning vectors with inserts obtained at step 3 or the collection of cells obtained after step 4 are called a gene library. This library is called a representative library if the sum of the genomic DNA inserts found in the cloning vectors of all cells represents the entire cellular DNA. Only a representative library, i.e., only the presence of all DNA fragments generated originally, guarantees a reasonable chance of finding the desired gene. Statistical analysis shows that for human DNA more than 600000 cell clones must be screened in order to find a particular DNA fragment containing the desired gene with a probability of 99 %. In principle libraries can be made also with cellular RNA as starting material. The resulting collection of host cells containing recombinant vectors is then called a cDNA library. The art of cloning is to find the one particular cell (marked red) which contains the cloning vector with the gene of interest. This process is called library screening. If this goal has been achieved the gene in question is said to have been cloned. Many different techniques are available for screening a library. The most important approaches involve screening by nucleic acid hybridization and screening by functional analysis. Nucleic acid hybridization requires some prior knowledge of the DNA sequence either of the gene to be cloned or of stretches of DNA in the vicinity of the gene to be cloned. Functional screening involves the use of expression vectors that allow cells containing the vector with the desired gene to express the corresponding protein. Under these circumstances, cells containing a vector with the desired gene can be identified by means of antibodies directed against the protein. Cells producing the desired gene product can also be identified in bioassays detecting protein activities, if these are available.

Many different techniques have been developed to isolate specific DNA clones from a library and if the process has been successful the specific DNA clone is said to have been cloned. Detection of an individual clone in a library can be achieved by employing strategies of nucleic acid hybridization in which short chemically synthesized labeled oligonucleotides are used to detect complementary sequences in individual cells or phages containing an insert. The sequences of such oligonucleotides used to identify the desired gene in the library can be derived, for example, from known protein sequences according to the rules of the genetic code. Related genes (see also: gene family) can be identified by altering the hybridization conditions (for example, low stringency hybridization, i.e., hybridization under conditions that allows base mismatches) and/or by using so-called degenerated oligonucleotides (mixtures of oligonucleotides that differ from each other by base substitutions at identical and/or different positions). The desired gene can be identified also by the activities of the encoded gene product. In this case, one uses a so-called expression library that have been established by cloning DNA fragments into special cloning vectors allowing the functional expression of cloned DNA fragments. Functional gene products and hence the desired clones can then be detected either by antibodies or other ligands that specifically recognize the encoded proteins or by exploiting a bioactivity of the gene product, if known.

In contrast to a genomic library which contains fragments of genomic DNA with intron and regulatory gene sequences a cDNA library contains inserts of cDNA fragments that correspond to the entire mRNAs of a cell. Such libraries therefore contain DNA fragments from which intron sequences have been removed. cDNA (or complementary DNA) is obtained by using the enzyme reverse transcriptase to copy mRNA sequences back into the corresponding DNA sequences. One of the advantages of a cDNA library is the improved frequency with which individual DNA fragments occur. This is due to the presence of multiple copies of a mRNA as opposed to DNA for any given gene. In addition, the representation of inserts derived from functionally expressed mRNAs found in a cDNA library also reflects the functional activities of the cells from which the library was established. Moreover, as cDNA clones are derived from mRNA, they do not contain any intervening sequences (introns). cDNA clones can be used, therefore, directly to express the proteins encoded by them (see also: gene expression, Recombinant cytokines). Many genes encoding cytokines have been isolated originally in the form of cDNA clones.

A special form a gene library that is gaining importance is the subtractive library or differential library. Terms such as subtractive hybridization, subtraction cloning, differential gene screening, differential hybridization, or genomic difference cloning refer to the screening of such a library with the aim to isolate a specific gene.

General strategy for subtractive library screening. Consider two cell types, designated A and B. These cells may be completely different (for example brain and liver cells) or identical cells that differ from each other, for example, in their activation state or pretreatment with cytokines. In the initial step (step 1) polyadenylated mRNA (jagged lines ending in AAAAA) is isolated from both types of cells. The RNA shown in green is expressed in cell type A only while that shown in blue is expressed in cell type B only. RNAs expressed in either cell type are shown in black. RNA isolated from cell type A is used to generate single-stranded cDNA by reverse transcription (step 2). This cDNA is then hybridized with RNA from cell type B (step 3). Those RNAs (black) expressed in both cell types will form DNA/RNA hybrids while cDNA single strands derived from type A-specific mRNA not present in type B-cells will remain single stranded. Likewise, RNA that is expressed in type B but not in type A cells will remain single stranded. Single and double stranded molecules are separated by hydroxyapatite chromatography (step 4). This yields fractions containing only single stranded DNA or RNA molecules. Removal of RNA by treatment with alkali yields a population of cDNA molecules derived from those RNAs expressed specifically in type A cells only. These can be converted into double stranded DNA which can then be cloned. If the aim is to investigate those RNAs expressed specifically in type B-cells only type B-specific RNA is converted into single stranded DNA and hybridized with RNA from type A cells.

Use of a subtractive library allows cloning of genes that are expressed differentially in two different cell types, i.e., genes expressed in a cell type-specific manner. These procedures can be used also to clone genes that are expressed differentially in response to environmental stimuli in the same type of cells.

The major advantage of subtractive libraries over other libraries lies in the fact that it allows cloning of genes without any previous knowledge of their sequences or functions (normally used as a criterion either for protein purification or for cDNA isolation in expression libraries), the only essential parameter being a difference in gene expression in the two types of cells being compared. Use of subtractive libraries has been instrumental in identifying new cytokine and cytokine receptor genes as well as a plethora of genes the expression of which is specifically induced by treatment of cells with cytokines or other stimuli (see also: Early response gene).

Technical advances, in particular the development of various strategies based upon the application of PCR amplification (for instance differential display PCR) have made screening of gene libraries and subtractive libraries either considerably easier or have even replaced classical library screening involving nucleic acid hybridization.

See REFERENCES for entry Gene library

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