9.1 - Genetic Analysis of Mutations to Identify and Study Genes

• The information encoded in the DNA sequence of genes specifies the sequence and therefore the structure and function of every protein molecule in a cell

• The power of genetics as a tool for studying cells and organisms lies in the ability of researchers to selectively alter every copy of just one type of protein in a cell by making a change in the gene for that protein.

• The different forms, or variants, of a gene, are referred to as alleles. Geneticists commonly refer to the numerous naturally occurring genetic variants that exist in populations, particularly human populations, as alleles

Recessive and Dominant Mutant Alleles Generally Have Opposite Effects on Gene Function

• A fundamental genetic difference between experimental organisms is whether their cells carry a single set of chromosomes or two copies of each chromosome

• The former are referred to as haploid; the latter, as diploid

• Since diploid organisms carry two copies of each gene, they may carry identical alleles, that is, be homozygous for a gene, or carry different alleles, that is, be heterozygous for a gene

• Recessive alleles usually result from a mutation that inactivates the affected gene, leading to a partial or complete loss of function

• Dominant mutations in certain genes are associated with a loss of function

Segregation of Mutations in Breeding Experiments Reveals Their Dominance or Recessivity

• Geneticists exploit the normal life cycle of an organism to test for the dominance or recessivity of alleles

• Like somatic cells, premeiotic germ cells are diploid, containing two homologs of each morphologic type of chromosome

• The two homologs constituting each pair of homologous chromosomes are descended from different parents, and thus their genes may exist in different allelic forms.

• Geneticists usually strive to begin breeding experiments with strains that are homozygous for the genes under examination

• In such true-breeding strains, every individual will receive the same allele from each parent and therefore the composition of alleles will not change from one generation to the next

Conditional Mutations Can Be Used to Study Essential Genes in Yeast

• The procedures used to identify and isolate mutants, referred to as genetic screens, depend on whether the experimental organism is haploid or diploid and, if the latter, whether the mutation is recessive or dominant

• Genes that encode proteins essential for life are among the most interesting and important ones to study

• In haploid yeast cells, essential genes can be studied through the use of conditional mutations

• Among the most common conditional mutations are temperature-sensitive mutations, which can be isolated in bacteria and lower eukaryotes but not in warm-blooded eukaryotes

• Once temperature-sensitive mutants were isolated, further analysis revealed that they indeed were defective in cell division

• In S. cerevisiae, cell division occurs through a bidding process, and the size of the bud, which is easily visualized by light microscopy, indicates a cell’s position in the cell cycle.

Recessive Lethal Mutations in Diploids Can Be Identified by Inbreeding and Maintained in Heterozygotes

• In diploid organisms, phenotypes resulting from recessive mutations can be observed only in individuals homozygous for the mutant alleles

• Since mutagenesis in a diploid organism typically changes only one allele of a gene, yielding heterozygous mutants, genetic screens must include inbreeding steps to generate progeny that are homozygous for the mutant alleles

Complementation Tests Determine Whether Different Recessive Mutations Are in the Same Gene

• In the genetic approach to studying a particular cellular process, researchers often isolate multiple recessive mutations that produce the same phenotype

• A common test for determining whether these mutations are in the same gene or in different genes exploits the phenomenon of genetic complementation, that is, the restoration of the wild-type phenotype by the mating of two different mutants

• Complementation analysis of a set of mutants exhibiting the same phenotype can distinguish the individual genes in a set of functionally related genes, all of which must function to produce a given phenotypic trait

Double Mutants Are Useful in Assessing the Order in Which Proteins Function

• Based on careful analysis of mutant phenotypes associated with a particular cellular process, researchers often can deduce the order in which a set of genes and their protein products function

• Ordering of Biosynthetic Pathways: A simple example of the first type of process is the biosynthesis of a metabolite such as the amino acid tryptophan in bacteria

• Ordering of Signaling Pathways: The expression of many eukaryotic genes is regulated by signaling pathways that are initiated by extracellular hormones, growth factors, or other signals

• Such signaling pathways may include numerous components, and double-mutant analysis often can provide insight into the functions and interactions of these components

• The only prerequisite for obtaining useful information from this type of analysis is that the two mutations must have opposite effects on the output of the same regulated pathway

Genetic Suppression and Synthetic Lethality Can Reveal Interacting or Redundant Proteins

• Two other types of genetic analysis can provide additional clues about how proteins that function in the same cellular process may interact with one another in the living cell

• Suppressor Mutations: The first type of analysis is based on genetic suppression

• To understand this phenomenon, suppose that point mutations lead to structural changes in one protein (A) that disrupt its ability to associate with another protein (B) involved in the same cellular process

• Similarly, mutations in protein B lead to small structural changes that inhibit its ability to interact with protein A

• Synthetic Lethal Mutations: A phenomenon, called synthetic lethality, produces a phenotypic effect opposite to that of suppression

• In this case, the deleterious effect of one mutation is greatly exacerbated (rather than suppressed) by a second mutation in the same or a related gene

• One situation in which such synthetic lethal mutations can occur

9.2 - DNA Cloning by Recombinant DNA Methods

• A variety of techniques often referred to as recombinant DNA technology, are used in DNA cloning, which permits researchers to prepare large numbers of identical DNA molecules

• Recombinant DNA is simply any DNA molecule composed of sequences derived from different sources

Restriction Enzymes and DNA Ligases Allow Insertion of DNA Fragments into Cloning Vectors

• A major objective of DNA cloning is to obtain discrete, small regions of an organism’s DNA that constitute specific genes

• Only relatively small DNA molecules can be cloned in any of the available vectors

• Cutting DNA Molecules into Small Fragments: Restriction enzymes are endonucleases produced by bacteria that typically recognize specific 4- to 8-bp sequences, called restriction sites, and then cleave both DNA strands at this site

• Restriction sites commonly are short palindromic sequences; that is, the restriction-site sequence is the same on each DNA strand when read in the 5’ → 3’ direction

• Many restriction enzymes make staggered cuts in the two DNA strands at their recognition site, generating fragments that have a single-stranded “tail” at both ends

• Inserting DNA Fragments into Vectors: DNA fragments with either sticky ends or blunt ends can be inserted into vector DNA with the aid of DNA ligases

• During normal DNA replication, DNA ligase catalyzes the end-to-end joining (ligation) of short fragments of DNA, called Okazaki fragments

E. coli Plasmid Vectors Are Suitable for Cloning Isolated DNA Fragments

• Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s chromosomal DNA

• These extrachromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g., yeast), exist in a parasitic or symbiotic relationship with their host cell

• The plasmids most commonly used in recombinant DNA technology are those that replicate in E. coli. Investigators have engineered these plasmids to optimize their use as vectors in DNA cloning

• DNA fragments from a few base pairs up to ≈20 kb commonly are inserted into plasmid vectors

• If special precautions are taken to avoid manipulations that might mechanically break DNA, even longer DNA fragments can be inserted into a plasmid vector

• When a recombinant plasmid with an inserted DNA fragment transforms an E. coli cell, all the antibiotic-resistant progeny cells that arise from the initially transformed cell will contain plasmids with the same inserted DNA

Bacteriophage λ Vectors Permit Efficient Construction of Large DNA Libraries

• Vectors constructed from bacteriophage are about a thousand times more efficient than plasmid vectors in cloning large numbers of DNA fragments

  • Phage vectors have been widely used to generate DNA libraries, comprehensive collections of DNA fragments representing the genome or expressed mRNAs of an organism

• A λ virion consists of a head, which contains the phage DNA genome, and a tail, which functions in infecting E. coli host cells

• The λ genes encoding the head and tail proteins, as well as various proteins involved in phage DNA replication and cell lysis, are grouped in discrete regions of the ≈50-kb viral genome

• It is technically feasible to use λ phage cloning vectors to generate a genomic library, that is, a collection of λ clones that collectively represent all the DNA sequences in the genome of a particular organism

cDNAs Prepared by Reverse Transcription of Cellular mRNAs Can Be Cloned to Generate cDNA Libraries

• The first step in preparing a cDNA library is to isolate the total mRNA from the cell type or tissue of interest

  • Because of their poly(A) tails, mRNAs are easily separated from the much more prevalent rRNAs and tRNAs present in a cell extract by use of a column to which short strings of thymidylate (oligo-dTs) are linked to the matrix

• Each plaque arises from a single recombinant phage, all the progeny λ phages that develop are genetically identical and constitute a clone carrying a cDNA derived from a single mRNA; collectively they constitute a λ cDNA library

• One feature of cDNA libraries arises because different genes are transcribed at very different rates

DNA Libraries Can Be Screened by Hybridization to an Oligonucleotide Probe

• Both genomic and cDNA libraries of various organisms contain hundreds of thousands to upwards of a million individual clones in the case of higher eukaryotes

• The basis for screening with oligonucleotide probes is the hybridization

  • The ability of complementary single-stranded DNA or RNA molecules to associate (hybridize) specifically with each other via base pairing

Oligonucleotide Probes Are Designed Based on Partial Protein Sequences

• Clearly, the identification of specific clones by the membrane-hybridization technique depends on the availability of complementary radiolabeled probes

• For an oligonucleotide to be useful as a probe, it must be long enough for its sequence to occur uniquely in the clone of interest and not in any other clones

• Chemical synthesis of single-stranded DNA probes of defined sequence can be accomplished by the series of reactions

Yeast Genomic Libraries Can Be Constructed with Shuttle Vectors and Screened by Functional Complementation

• In some cases, a DNA library can be screened for the ability to express a functional protein that complements a recessive mutation

• Such a screening strategy would be an efficient way to isolate a cloned gene that corresponds to an interesting recessive mutation identified in an experimental organism

• Libraries constructed for the purpose of screening among yeast gene sequences usually are constructed from genomic DNA rather than cDNA

• To increase the probability that all regions of the yeast genome are successfully cloned and represented in the plasmid library

  • The genomic DNA usually is only partially digested to yield overlapping restriction fragments of ≈10 kb

9.3 - Characterizing and Using Cloned DNA Fragments

Gel Electrophoresis Allows Separation of Vector DNA from Cloned Fragments

• In order to manipulate or sequence a cloned DNA fragment, it first must be separated from the vector DNA

• This can be accomplished by cutting the recombinant DNA clone with the same restriction enzyme used to produce the recombinant vectors originally

• Near neutral pH, DNA molecules carry a large negative charge and therefore move toward the positive electrode during gel electrophoresis

• A common method for visualizing separated DNA bands on a gel is to incubate the gel in a solution containing the fluorescent dye ethidium bromide

• This planar molecule binds to DNA by intercalating between the base pairs

Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method

• The complete characterization of any cloned DNA fragment requires the determination of its nucleotide sequence. F. Sanger and his colleagues developed the method

  • Now most commonly used to determine the exact nucleotide sequence of DNA fragments up to ≈500 nucleotides long

The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture

• If the nucleotide sequences at the ends of a particular DNA region are known, the intervening fragment can be amplified directly by the polymerase chain reaction (PCR)

• The PCR depends on the ability to alternately denature (melt) double-stranded DNA molecules and renature (anneal) complementary single strands in a controlled fashion

• Direct Isolation of a Specific Segment of Genomic DNA: For organisms in which all or most of the genome has been sequenced, PCR amplification starting with the total genomic DNA often is the easiest way to obtain a specific DNA region of interest for cloning

• Preparation of Probes: Preparation of such probes by PCR amplification requires chemical synthesis of only two relatively short primers corresponding to the two ends of the target sequence

• Tagging of Genes by Insertion Mutations: Another useful application of the PCR is to amplify a “tagged” gene from the genomic DNA of a mutant strain

• This approach is a simpler method for identifying genes associated with a particular mutant phenotype than the screening of a library by functional complementation

• The key to this use of PCR is the ability to produce mutations by insertion of a known DNA sequence into the genome of an experimental organism

Blotting Techniques Permit Detection of Specific DNA Fragments and mRNAs with DNA Probes

• Two very sensitive methods for detecting a particular DNA or RNA sequence within a complex mixture combine separation by gel electrophoresis and hybridization with a complementary radiolabeled DNA probe

• Southern Blotting The first blotting technique to be devised is known as Southern blotting after its originator E. M. Southern

  • This technique is capable of detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavage of the entire human genome with a restriction enzyme

• Northern Blotting One of the most basic ways to characterize a cloned gene is to determine when and where in an organism the gene is expressed

  • Expression of a particular gene can be followed by assaying for the corresponding mRNA by Northern blotting, named, in a play on words, after the related method of Southern blotting

E. coli Expression Systems Can Produce Large Quantities of Proteins from Cloned Genes

• The first step in producing large amounts of a low-abundance protein is to obtain a cDNA clone encoding the full-length protein by methods discussed previously

• The second step is to engineer plasmid vectors that will express large amounts of the encoded protein when it is inserted into E. coli cells

Plasmid Expression Vectors Can Be Designed for Use in Animal Cells

• One disadvantage of bacterial expression systems is that many eukaryotic proteins undergo various modifications (e.g., glycosylation, hydroxylation) after their synthesis on ribosomes

• To get around this limitation, cloned genes are introduced into cultured animal cells, a process called transfection

• Two common methods for transfecting animal cells differ in whether the recombinant vector DNA is or is not integrated into the host-cell genomic DNA

• In both methods, cultured animal cells must be treated to facilitate their initial uptake of a recombinant plasmid vector

• Transient Transfection: The simplest of the two expression methods, called transient transfection, employs a vector similar to the yeast shuttle vectors described previously

• Stable Transfection (Transformation) If an introduced vector integrates into the genome of the host cell, the genome is permanently altered and the cell is said to be transformed

• Integration most likely is accomplished by mammalian enzymes that function normally in DNA repair and recombination

• Epitope Tagging: In addition to their use in producing proteins that are modified after translation, eukaryotic expression vectors provide an easy way to study the intracellular localization of eukaryotic proteins

  • In this method, a cloned cDNA is modified by fusing it to a short DNA sequence encoding an amino acid sequence recognized by a known monoclonal antibody

9.4 - Genomics: Genome-wide Analysis of Gene Structure and Expression

Stored Sequences Suggest Functions of Newly Identified Genes and Proteins

• Proteins with similar functions often contain similar amino acid sequences that correspond to important functional domains in the three-dimensional structure of the proteins

• By comparing the amino acid sequence of the protein encoded by a newly cloned gene with the sequences of proteins of known function, an investigator can look for sequence similarities that provide clues to the function of the encoded protein

• Even when a protein shows no significant similarity to other proteins with the BLAST algorithm, it may nevertheless share a short sequence with other proteins that are functionally important

• Such short segments recurring in many different proteins referred to as motifs, generally have similar functions

Comparison of Related Sequences from Different Species Can Give Clues to Evolutionary Relationships Among Proteins

• BLAST searches for related protein sequences may reveal that proteins belong to a protein family. (The corresponding genes constitute a gene family)

• Protein families are thought to arise by two different evolutionary processes, gene duplication, and speciation

• All the different members of the tubulin family are sufficiently similar in sequence to suggest a common ancestral sequence

• All these sequences are considered to be homologous

• More specifically, sequences that presumably diverged as a result of gene duplication (e.g., the - and-tubulin sequences) are described as paralogous

Genes Can Be Identified Within Genomic DNA Sequences

• The complete genomic sequence of an organism contains within it the information needed to deduce the sequence of every protein made by the cells of that organism

• For organisms such as bacteria and yeast, whose genomes have few introns and short intergenic regions

  • Most protein-coding sequences can be found simply by scanning the genomic sequence for open reading frames (ORFs) of significant length

• The best gene-finding algorithms combine all the available data that might suggest the presence of a gene at a particular genomic site

The Size of an Organism’s Genome Is Not Directly Related to Its Biological Complexity

• The combination of genomic sequencing and gene-finding computer algorithms has yielded the complete inventory of protein-coding genes for a variety of organisms

• The functions of about half the proteins encoded in these genomes are known or have been predicted on the basis of sequence comparisons

• One of the surprising features of this comparison is that the number of protein-coding genes within different organisms does not seem proportional to our intuitive sense of their biological complexity

DNA Microarrays Can Be Used to Evaluate the Expression of Many Genes at One Time

• Monitoring the expression of thousands of genes simultaneously is possible with DNA microarray analysis

• A DNA microarray consists of thousands of individual, closely packed gene-specific sequences attached to the surface of a glass microscopic slide

• Preparation of DNA Microarrays In one method for preparing microarrays, a ≈1-kb portion of the coding region of each gene analyzed is individually amplified by PCR

  • In an alternative method, multiple DNA oligonucleotides, usually at least 20 nucleotides in length, are synthesized from an initial nucleotide that is covalently bound to the surface of a glass slide

  • The synthesis of an oligonucleotide of a specific sequence can be programmed in a small region on the surface of the slide

• Effect of Carbon Source on Gene Expression in Yeast: The initial step in a microarray expression study is to prepare fluorescently labeled cDNAs corresponding to the mRNAs expressed by the cells understudy

Cluster Analysis of Multiple Expression Experiments Identifies Co-regulated Genes

• Firm conclusions rarely can be drawn from a single microarray experiment about whether genes that exhibit similar changes in expression are co-regulated and hence likely to be closely related functionally

• Genes that appear to be co-regulated in a single microarray expression experiment may undergo changes in expression for very different reasons and may actually have very different biological functions

• A solution to this problem is to combine the information from a set of expression array experiments to find genes that are similarly regulated under a variety of conditions or over a period of time

9.5 - Inactivating the Function of Specific Genes in Eukaryotes

• The elucidation of DNA and protein sequences in recent years has led to the identification of many genes, using sequence patterns in genomic DNA and the sequence similarity of the encoded proteins with proteins of known function

Normal Yeast Genes Can Be Replaced with Mutant Alleles by Homologous Recombination

• Modifying the genome of the yeast Saccharomyces is particularly easy for two reasons: yeast cells readily take up exogenous DNA under certain conditions, and the introduced DNA is efficiently exchanged for the homologous chromosomal site in the recipient cell

• This specific, targeted recombination of identical stretches of DNA allows any gene in yeast chromosomes to be replaced with a mutant allele

• Disruption of yeast genes by this method is proving particularly useful in assessing the role of proteins identified by ORF analysis of the entire genomic DNA sequence

• A large consortium of scientists has replaced each of the approximately 6000 genes identified by ORF analysis with the kanMX disruption construct and determined which gene disruptions lead to nonviable haploid spores

Transcription of Genes Ligated to a Regulated Promoter Can Be Controlled Experimentally

• Although disruption of an essential gene required for cell growth will yield non-viable spores

  • This method provides little information about what the encoded protein actually does in cells

• A useful promoter for this purpose is the yeast GAL1 promoter, which is active in cells grown on galactose but completely inactive in cells grown on glucose

• In this approach, the coding sequence of an essential gene (X) ligated to the GAL1 promoter is inserted into a yeast shuttle vector

• In an early application of this method, researchers explored the function of cytosolic Hsc70 genes in yeast

• Haploid cells with a disruption in all four redundant Hsc70 genes were non-viable unless the cells carried a vector containing a copy of the Hsc70 gene that could be expressed from the GAL1 promoter on galactose medium

Specific Genes Can Be Permanently Inactivated in the Germ Line of Mice

• Many of the methods for disrupting genes in yeast can be applied to genes of higher eukaryotes

• These genes can be introduced into the germline via homologous recombination to produce animals with a gene knockout, or simply “knockout”

• Gene-targeted knockout mice are generated by a two-stage procedure

  • In the first stage, a DNA construct containing a disrupted allele of a particular target gene is introduced into embryonic stem (ES) cells

    • These cells, which are derived from the blastocyst, can be grown in culture through many generations

  • In the second stage in the production of knockout mice, ES cells heterozygous for a knockout mutation in gene X are injected into a recipient wild-type mouse blastocyst, which subsequently is transferred into a surrogate pseudopregnant female mouse

Somatic Cell Recombination Can Inactivate Genes in Specific Tissues

• Investigators often are interested in examining the effects of knockout mutations in a particular tissue of the mouse, at a specific stage in development, or both

• Mice carrying a germ-line knockout may have defects in numerous tissues or die before the developmental stage of interest

  • To address this problem, mouse geneticists have devised a clever technique to inactivate target genes in specific types of somatic cells or at particular times during development

  • This technique employs site-specific DNA recombination sites (called loxP sites) and the enzyme Cre that catalyzes recombination between them

  • The loxP-Cre recombination system is derived from bacteriophage P1, but this site-specific recombination system also functions when placed in mouse cells

Dominant-Negative Alleles Can Functionally Inhibit Some Genes

• For certain genes, the difficulties in producing homozygous knockout mutants can be avoided by the use of an allele carrying a dominant-negative mutation

• These alleles are genetically dominant; that is, they produce a mutant phenotype even in cells carrying a wild-type copy of the gene

• But unlike other types of dominant alleles, dominant-negative alleles produce a phenotype equivalent to that of a loss-of-function mutation

• Useful dominant-negative alleles have been identified for a variety of genes and can be introduced into cultured cells by transfection or into the germline of mice or other organisms

Double-Stranded RNA Molecules Can Interfere with Gene Function by Targeting mRNA for Destruction

• Researchers are exploiting a recently discovered phenomenon known as RNA interference (RNAi) to inhibit the function of specific genes

• This approach is technically simpler than the methods described above for disrupting genes

• To use RNAi for intentional silencing of a gene of interest, investigators first produce dsRNA based on the sequence of the gene to be inactivated

• This dsRNA is injected into the gonad of an adult worm, where it has access to the developing embryos

• As the embryos develop, the mRNA molecules corresponding to the injected dsRNA are rapidly destroyed

• The resulting worms display a phenotype similar to the one that would result from disruption of the corresponding gene itself

• Initially, the phenomenon of RNAi was quite mysterious to geneticists. Recent studies have shown that specialized RNA-processing enzymes cleave dsRNA into short segments, which base-pair with endogenous mRNA

9.6 - Identifying and Locating Human Disease Genes

• Inherited human diseases are the phenotypic consequence of defective human genes

• Although a “disease” gene may result from a new mutation that arose in the preceding generation, most cases of inherited diseases are caused by preexisting mutant alleles that have been passed from one generation to the next for many generations

• The genes responsible for inherited diseases must be found without any prior knowledge or reasonable hypotheses about the nature of the affected gene or its encoded protein

Many Inherited Diseases Show One of Three Major Patterns of Inheritance

• Human genetic diseases that result from a mutation in one specific gene exhibit several inheritance patterns depending on the nature and chromosomal location of the alleles that cause them

• One characteristic pattern is that exhibited by a dominant allele in an autosome (that is, one of the 22 human chromosomes that are not a sex chromosome)

• A recessive allele in an autosome exhibits a quite different segregation pattern. For an autosomal recessive allele, both parents must be heterozygous carriers of the allele in order for their children to be at risk of being affected by the disease

Recombinational Analysis Can Position Genes on a Chromosome

• The independent segregation of chromosomes during meiosis provides the basis for determining whether genes are on the same or different chromosomes

• Genetic traits that segregate together during meiosis more frequently than expected from random segregation are controlled by genes located on the same chromosome

• The presence of many different already mapped genetic traits, or markers, distributed along the length of a chromosome facilitates the mapping of a new mutation by assessing its possible linkage to these marker genes in appropriate crosses

• The more markers that are available, the more precisely a mutation can be mapped

DNA Polymorphisms Are Used in Linkage-Mapping Human Mutations

• Many different genetic markers are needed to construct a high-resolution genetic map

• In the experimental organisms commonly used in genetic studies, numerous markers with easily detectable phenotypes are readily available for genetic mapping of mutations

• Restriction fragment length polymorphisms (RFLPs) were the first type of molecular markers used in linkage studies

• RFLPs arise because mutations can create or destroy the sites recognized by specific restriction enzymes, leading to variations between individuals in the length of restriction fragments produced from identical regions of the genome

Linkage Studies Can Map Disease Genes with a Resolution of About 1 Centimorgan

• How the allele conferring a particular dominant trait (e.g., familial hypercholesterolemia) might be mapped

  • The first step is to obtain DNA samples from all the members of a family containing individuals that exhibit the disease

  • The DNA from each affected and unaffected individual then is analyzed to determine the identity of a large number of known DNA polymorphisms (either SSR or SNP markers can be used)

  • The segregation pattern of each DNA polymorphism within the family is then compared with the segregation of the disease under study to find those polymorphisms that tend to segregate along with the disease

  • Lastly, computer analysis of the segregation data is used to calculate the likelihood of linkage between each DNA polymorphism and the disease-causing allele

  • A phenomenon called linkage disequilibrium is the basis for an alternative strategy, which in some cases can afford a higher degree of resolution in mapping studies

Further Analysis Is Needed to Locate a Disease Gene in Cloned DNA

• Although linkage mapping can usually locate a human disease gene to a region containing about 7.5 x 105 base pairs, as many as 50 different genes may be located in a region of this size

• The ultimate objective of a mapping study is to locate the gene within a cloned segment of DNA and then to determine the nucleotide sequence of this fragment

• In many cases, point mutations that give rise to disease-causing alleles may result in no detectable change in the level of expression or electrophoretic mobility of mRNAs

• So if the comparison of the mRNAs expressed in normal and affected individuals reveals no detectable differences in the candidate mRNAs, a search for point mutations in the DNA regions encoding the mRNAs is undertaken

Many Inherited Diseases Result from Multiple Genetic Defects

• Most of the inherited human diseases that are now understood at the molecular level are monogenetic traits

• That is, a clearly discernible disease state is produced by the presence of a defect in a single gene

• Monogenic diseases caused by a mutation in one specific gene exhibit

• Many other inherited diseases show more complicated patterns of inheritance, making the identification of the underlying genetic cause much more difficult

• Human geneticists used two different approaches to identify the many genes associated with retinitis pigmentosa

• A further complication in the genetic dissection of human diseases is posed by diabetes, heart disease, obesity, predisposition to cancer, and a variety of mental disorders that have at least some heritable properties

• Models of human disease in experimental organisms may also contribute to unraveling the genetics of complex traits such as obesity or diabetes