Gene library

The unit of heredity most simply defined as a specific segment of DNA, usually in the order of 1000 nucleotides, that specifies a single polypeptide. Many phenotypic characteristics are determined by a single gene, while others are multigenic. Genes are specifically located in linear order along the single DNA molecule that makes up each chromosome. All eukaryotic cells contain a diploid (2n) set of chromosomes so that two copies of each gene, one derived from each parent, are present in each cell; the two copies often specify a different phenotype, i.e. the polypeptide will have a somewhat different amino acid composition. These alternative forms of gene, both within and between individuals, are called alleles. Genes determine the physical (structural genes), the biochemical (enzymes), physiological and behavioral characteristics of an animal. The formation of gametes (sperm, ova) involves a process of meiosis, which allows crossing over between four pairs of chromosomes, two derived from each parent, which means that new forms of a particular chromosome are created. Gamete formation also results in cells (gametes) with a haploid (n) set of chromosomes that in fertilization creates a new individual, which is a recombinant of 2n chromosomes, half derived by way of the ovum from the mother and half via the spermatozoa from the father. Changes in the nucleotide sequence of a gene, either by substitution of a different nucleotide or by deletion or insertion of other nucleotides, constitute mutations which add to the diversity of animal species by creating different alleles and can be used as a basis for genetic selection of different phenotypes. Some mutations, be they a single base change in a single gene or a major deletion, are lethal. gene action the way in which genes exert their effects on tissues or processes, e.g. by being dominant or recessive, or partially so, being absent, being sex-linked, being involved in chromosomal aberrations. allelic g's different forms of a particular gene usually situated at the same position (locus) in a pair of chromosomes. gene amplification see gene duplication (below). gene bank the collection of DNA sequences in a given genome. Called also gene library.

barring gene responsible for the barred pattern on the feathers of Barred Plymouth Rock birds. gene box gene clone gene cluster a group of related genes derived from a common ancestral gene, located closely together on the same chromosome. Called also multigene family. complementary g's two independent pairs of nonallelic genes, neither of which is functional without the other. gene conversion a non-reciprocal exchange of DNA elements during meiosis which results in a functional rearrangement of chromosomal DNA. dhfr gene dihydrofolate reductase gene; an enzyme required to maintain cellular concentrations of H2 folate for nucleotide biosynthesis, and which has been used as a 'selective marker'; cells lacking the enzyme only survive in media containing thymidine, glycine and purines; mutant cells (dhfr) transfected with DNA that is dhfr can be selectively grown in medium lacking these elements. diversity (D) gene genes located in diversity (D) segment; contribute to the hypervariable region of immunoglobulins. dominant gene one that produces an effect (the phenotype) in the organism regardless of the state of the corresponding allele. Examples of traits determined by dominant genes are short hair in cats and black coat color in dogs. gene duplication as a result of non-homologous recombination, a chromosome carries two or more copies of a gene. gene expression

see expression (3). gene frequency the proportion of the substances or animals in the group which carry a particular gene. holandric g's genes located on the Y chromosome and appearing only in male offspring. immune response (Ir) g's genes of the major histocompatibility complex (MHC) that govern the immune response to individual immunogens. jumping gene see mobile dna. gene knockout replacement of a normal gene with a mutant allele, as in gene knockout mice. lethal gene one whose presence brings about the death of the organism or permits survival only under certain conditions. gene library gene locus mutant gene one that has undergone a detectable mutation. non-protein encoding gene the final products of some genes are RNA molecules rather than proteins. overlapping g's when more than one mRNA is transcribed from the same DNA sequence; the mRNAs may be in the same reading frame but of different size or they may be in different reading frames. gene pool total of all genes possessed by all members of the population which are capable of reproducing during their lifetime.

gene probe recessive gene one that produces an effect in the organism only when it is transmitted by both parents, i.e. only when the individual is homozygous. regulator gene, repressor gene one that synthesizes repressor, a substance which, through interaction with the operator gene, switches off the activity of the structural genes associated with it in the operon. reporter gene one that produces products which can be measured and therefore used as an indicator of whether a DNA construct has successfully been transferred. sex-linked gene one that is carried on a sex chromosome, especially an X chromosome. gene splicing structural gene nucleotide sequences coding for proteins. gene therapy the insertion of functional genes into cells of the host in order to alter its phenotype, usually used to treat an inherited defect. gene transcription gene transfer tumor suppressor g's a class of genes that encode proteins that normally suppress cell division that when mutated allow cells to continue unrestricted cell division and may result in a tumor. Saunders Comprehensive Veterinary Dictionary, 3 ed. 2007 Elsevier, Inc. All rights reserved gene library Genomic library Genetics A molecular 'database' created when the mRNA extracted from a given tissue is reverse transcribed into cDNA (complementary DNA) segments, which in toto comprise the sequences of genes expressed in the tissue of interest. See Library Molecular

biology A random collection of DNA fragmentstypically representing the entire genome of an organism that have been inserted into a cloning vector.

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 Bspecific 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.

Human genomic libraries can be constructed using restriction nucleases and ligase. A genomic library comprises a set of bacteria, each carrying a different small fragment of human DNA. For simplicity, cloning of just a few representative fragments (colored) is shown. In reality, all the gray DNA fragments will also be cloned.