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Introduction
The bovine diploid genome consists of approximately 6000 million base pairs of DNA, distributed over 29 pairs of acrocentric autosomes and a pair of sex chromosomes. It is estimated that between 50,000 and 100,000 pairs of genes are encoded in the bovine genome, of which about 300 have been mapped to chromosomes (1). The average bovine chromosome is approximately 100 million base pairs long containing 2,000 to 5,000 genes which consist of coding sequences, known as exons, interrupted by intervening sequences referred to as introns. The non-coding regions of DNA are often repetitive sequences and are generally very polymorphic. It has been estimated that the genomes from two individuals vary by at least one nucleotide in every 300-1,000 bp on average (2). A large proportion of these sequence variants (30-40%) have been shown to occur as single base changes (3) or as variations in the number and type of repeat sequences. It is these polymorphisms that form the molecular basis of DNA genotyping.
Until 1988, analysis of DNA polymorphisms was essentially limited to the characterization of restriction fragment length polymorphism (RFLP) (4). With the polymerase chain reaction (PCR), analysis of polymorphisms based on length variation in tandemly repeated DNA became popular (5). These polymorphisms are termed variable number of tandem repeats (VNTRs). Nearly 23 percent of the genome in higher animals consists of repetitive sequence of DNA identified as micro, midi and macro satellites (6).
Restriction enzymes recognize specific sequences in the DNA and cause endonucleolytic cleavage where sites occur across the genome producing fragments of varying length (7). RFLPs occur frequently, follow Mendelian inheritance, and usually have a limited number of alleles with low heterozygosity and polymorphic information content. Relatively large amounts of DNA are required for detecting RFLPs and involve laborious technique. Hence RFLPs are not suitable for routine typing (8).
VNTRs are sequences that occur repeatedly throughout the genome and are highly polymorphic, due to variation in the number of repeats at a particular locus. (9). VNTR loci are divided into two classes: minisatellites and microsatellites. The difference between these markers are in the length and size of the individual repeat units: 15-60 bp for minisatellites (9) and 2-5bp for microsatellites (10). In cattle, minisatellites are clustered in the telomeres of chromosomes (11) whereas microsatellites appear to be distributed throughout the genome (12). Another major difference between these loci is the method of genotyping. Minisatellites are analysed by Southern blotting in the same manner as for RFLPs, but microsatellites are characterized by PCR amplification and polyacrylamide gel electrophoresis.
Even though various other types of DNA markers have been detected such as Randomly Amplified Polymorphic DNA (RAPD), Short interspersed nuclear elements (SINE or Alu repeats) and Amplified Fragment Length Polymorphism (AFLP), microsatellite markers overcome the difficulties of these marker types and are versatile tools for genome mapping. Thus these markers have proved to be of great use for studies in population and ecological genetics, gene mapping and medical genetics and are currently the favoured markers in human and animal genetic research.
Genetic markers and analysis of diseases
Congenital abnormalities are defects in the structure or function evident at birth, not necessarily caused by defective genes. Environmental influence that interferes with normal developmental processes may cause the observed defects. Inherited diseases may be congenital, but most of the known genetic diseases do not become evident for months or even years. Without a programme for screening for the disorder, carrier status often becomes apparent when two individuals are crossed, producing affected offspring. Carrier testing and selective breeding schemes will help breeders to eliminate the disease allele from the population.
The application of molecular biology has revolutionized our understanding of the genetic basis of disease susceptibility in both man and animals. Before the advent of DNA technology, geneticists used blood group and protein polymorphisms, visible cytogenetic aberrations (deletions, duplications, translocations) and other phenotypic markers to map hereditary diseases. For example, X-linked haemophilia was shown to be linked to red green colour blindness as early as 1937. By the 1980s it became possible to locate the genes responsible for diseases inherited in a Mendelian fashion to specific chromosomes by genome mapping techniques. The development of PCR has revolutionized the field of molecular biology and the detection of genetic disorders. Correlating the inheritance of highly polymorphic markers with diseases in families has enabled the mapping of mutations to specific chromosomal regions and discovery of candidate genes (13).
The identification of cystic fibrosis gene (14) has demonstrated the validity of “reverse genetics” approach (15) to genetic diseases. Reverse genetics has been identified as the application of linked markers to describe the chromosomal location of a gene involved in a disease, followed by efforts to identify and isolate the gene from its map position. Using this approach a large number of diseases have already been assigned to chromosomal regions (16).
To date four genetic disorders in animals known to be caused by point mutation have been localized e.g. hyperkalaemic periodic paralysis in quarter horses (HYPP) (17), porcine malignant hyperthermia (18), retinal atrophy in Irish setters (19) and deficiency of uridine monophosphate synthase (DUMPS) (20).
Different approaches are used for the identification of disease genes, viz. 1. cytogenetic approach, 2. linkage analysis, 3. genome mapping and 4. positional cloning.
1. Cytogenetic approach: Although in the early part of the century, cytogeneticists postulated that chromosomes provide hereditary basis for certain human diseases, it was not until 1959 that a chromosomal defect was established as the cause of a specific disease in man (21). Since then, a number of human syndromes such as Down’s syndrome, Turner’s syndrome and Klinefelter’s syndrome have been recognised as specific chromosome anomalies. In bovine, the cytogenetic approach has attracted attention through the discovery of chromosome alterations such as gonadal dysgenesis, freemartinism, 1/29 translocation and XX/XY chimerism. Chromosomal rearrangements as a cause of inherited diseases have not been as well studied as in man, but when found, they simplify the task of localizing the gene.
2. Linkage analysis: Linkage analysis using genetic markers has been the most powerful and practical tool in medical genetics for locating disease genes since the mid 1970s. The markers used for linkage analysis must be readily detectable and be found in a number of distinguishable variants throughout the population for correlating the inheritance of a marker and disease gene. Linked-markers provide the starting point for the identification and characterization of disease loci. Several diseases have been mapped to specific chromosomes by the method of linkage analysis in man e.g. Huntingtons disease and cystic fibrosis (22,23).
The method for analyzing linkage data is the lod score method or log of odds of probability for or against linkage. Linkage can be considered established when in a collection of families the sum of the lod scores at any value of recombination fraction (Ø) reaches +3, corresponding to odds for linkage of at least 1,000:1. If, however, the sum of the lod scores at a given value of (Ø) is less than –2, then the linkage is considered to be excluded.
Linked-markers may be several million base pairs of DNA distant from the disease gene. In order to bracket the area of chromosome to be searched, it is necessary to identify flanking polymorphic markers. Having identified a set of markers linked to the trait of interest, the linear order relative to each other, is determined by the frequency of recombination between them caused by crossover between the marker and the disease gene. Once the most plausible order for a cluster of linked markers has been established, they are assigned to specific chromosomes.
3. Genome mapping approach: Coverage of markers in human and mouse genomes is better than that in the bovine genome (1). The need for systematic analysis of the entire bovine genome and thus the construction of a gene map was discussed. Hence, the construction of a linkage map, a physical map and a comparative map is underway.
Linkage map is based on the proposal that a gene map with as few as 150 markers, evenly spaced, should provide a genetic marker every 20 cM along the genome. With markers spaced at 20 cM intervals, genes of interest would be no more than 10 cM from a marker and linkage will be detectable even in relatively small families (24). The genetic markers of choice are the microsatellite DNA markers because of the distribution over the entire genome and the ease of detecting polymorphism.
The physical map is simply the identification of the location of the genes (or marker loci) on chromosomes or on large DNA clones. Various physical mapping methods include somatic cell hybridization, and in situ hybridization. Comparative mapping entails the comparison of the organization of the genome to that of other species (25). Studies in man, mice, cattle, pigs and cats demonstrate that the arrangement of genes is roughly conserved through evolution with only modest rearrangements of chromosomes. The chromosomal localization of linked-markers can be achieved by various physical mapping methods. Once the chromosomal localization of the gene of interest has been determined, an inspection of the homologous regions of chromosome in man or mouse is made for any obvious candidate genes with physiological roles that are likely to be involved in the disease. If such candidate genes are found, the homologous ones are isolated from cDNA libraries. This candidate gene is screened for the difference in sequence between the affected and normal individuals. This is done by detecting polymorphisms in the different regions of the gene using techniques like denaturing gradient gel electrophoresis (DGGE) and single stranded conformation polymorphisms (SSCP) (26, 27) which can detect even single base pair changes. On detecting a change between the affected and unaffected group, that section of the gene is sequenced to identify the variation in the defective cells.
For many disorders no obvious candidate gene has been identified. In these cases, the mutation has to be localized first to a particular region of the chromosome, then genes in the region identified by a positional cloning approach.
4. Positional cloning: In the absence of candidate genes, the chromosomal regions identified by linkage analysis will be mapped at high resolution as a prelude to positional cloning. Initially, isolation and identification of a chromosome or region specific isolation of DNA fragments is done by somatic cell hybrid panels (28), chromosome sorting (29) or by micro-dissection of specific chromosome regions (30). Positional cloning of the gene is done without any information about its protein product. The inheritance of each marker is compared with that of the disorder in families or groups of families. The markers act as guides to chromosomal location, each marker flagging a short stretch of a chromosome. Isolation of adjacent or nearby DNA fragments in regions localized for disease gene is done by chromosome walking, by the use of a probe to identify additional cloned DNA that overlaps it, followed by preparation of probes from the farthest end of the new clone and re-screening to continue down the chromosome. Even though the technique in theory is simple, it is laborious to perform (31).
Detection of mutation in DNA
Demonstration of variation is a critical step in many studies in molecular genetics. In screening for mutations in genes that contribute to a particular disease process, direct gene analysis is the common strategy. Currently, mutations are detected by different procedures such as altered banding patterns of single-stranded DNA on non-denaturing gels (SSCP) (27), resolution of heteroduplex molecules by their instability in denaturing gradient gels (DGGE) (26), cleaving DNA with chemicals (32) or with ribonuclease A (33) or direct sequencing of the DNA segments.
DNA tests are employed to detect the carriers in the population. For instance, Bovine Leukocyte Adhesion Deficiency is a recessive disorder in Holstein cattle caused by point mutations within the gene encoding bovine alpha subunit of CD18 and carriers are identified by the PCR-RFLP (34). Similarly, DUMPS, a monogenic disorder in the Holstein breed has been shown to result from a point mutation generating a stop codon in UMPS. Carriers are detected by a direct DNA test based on PCR (20). HYPP in horses caused by cytosine to guanine substitution in transmembrane domain IVS3 of the alpha subunit of the muscle sodium channel gene is detected by a PCR-RFLP test (17).
Transgenic breeding strategies
In the current livestock breeding strategies, increasing the frequency of advantageous alleles of many loci brings about genetic improvement, though the actual loci are rarely identified. It has opened up many vistas in understanding behaviour and expression of a gene. Transgenic dairy animals have been developed for the production of pharmaceutical proteins in milk and animals with altered milk composition (35). In transgenesis, molecular markers serve as the reference points for mapping the relevant genes that would be the first step towards their identification, isolation, cloning, and their manipulation. After the successful production of transgenic animals, appropriate breeding methods could be followed for the multiplication of the herd.
Breeding and engineering animals.
The application of genetics has resulted in major improvements in both the productivity and well being of livestock and other animals. One of the most recent examples, announced early in 1993, was the development of a pig free of a particular mutation that increases the animal’s susceptibility to stress. Though linked with increased leanness of the meat, and positively selected in breeding for that reason, its undesirable consequences have led to the gene being bred out in a programme carried out at the Cotswold Pig Development Company in the U.K.
Now traditional animal breeding is being augmented by genetic engineering to make transgenic animals. The females are superovulated, providing a large supply of eggs. After fertilization, egg is immobilized under a microscope and cloned DNA is injected directly into one of its two pronuclei. Each pronucleus (one derived from the egg cell, the other from the spermatozoon) contains the haploid number of chromosomes. In a proportion of cases, the injected DNA integrates into one or more of the chromosomes. Manipulated offspring can then be screened for the presence of the introduced gene (36).
Another approach is finding increasing favour because it can position the new gene more precisely. DNA is transferred into embryonic stem cells - cells derived from pre-implantation embryos that can be grown indefinitely in the test tube. Cells that express the introduced DNA (i.e. they produce the relevant protein) can be selected in the test tube and be injected into a normal embryo and then be transferred into a foster mother. The immediate offsprings following this type of manipulation are chimeras, some of the cells in various organs (including the gonads) being genetically altered cells. However, breeding from this generation leads to segregation of the introduced gene, so that a proportion of the next generation consists of completely transgenic animals. The opportunity to manipulate and select cells in the test tube means that gene may, if the sequence is known, be targeted at a specific site in the chromosome (37).
Why make transgenic animals?
One purpose of genetic engineering is to improve animal health. Genes responsible for resistance to particular diseases might be introduced into otherwise vulnerable breeds of animals, to protect them against those conditions. One example is the genes conferring natural resistance to trypanosomiasis, which occurs in certain breeds of cattle but could be transferred into breeds that are desirable in otherways.
Another is to produce rare and expensive proteins for use in human medicine. The genes coding for certain proteins, for example, can be expressed in the sheep mammary gland, the protein being recovered from the animal’s milk. Researcher in Edinburgh, U.K, have already used this approach to arrange for sheep to secrete in their milk the blood clotting protein known as factor IX whose absence causes one type of haemophilia. When inserted into fertilized eggs the new gene alongside the regulatory region of the lactoglobulin gene, encodes the specific protein in sheep’s milk. Another protein produced in this way recently in sheep is a-1-antitrypsin (ATT). Humans normally make their own ATT, which checks the action of elastin, an enzyme that can otherwise attack lung tissue. ATT is required to treat patients with congenital emphysema, whose own ATT is either faulty or absent altogether (38).
Recent research with transgenic animals has pointed the way towards the treatment of human genetic disorders. Working with mouse stem cells, scientists in Edinburgh selected cells with a mutation in the gene for the production of the enzyme hypoxanthine phosphoribosyl transferase (HPRT). When these cells were introduced into embryos and transferred to foster mothers, some of the offsprings were totally deficient in HPRT. But the deficiency could be corrected by inserting an HPRT gene into the stem cells. This was the first demonstration that precise genetic alteration could be introduced into a mammal’s germ line. In humans, absence of HPRT results in the severe and usually fatal Lesch Nyhan syndrome. There are hopes, therefore, that this work could lead to methods of correcting this and similar conditions.
Making medicines by genetic engineering
Genetically modified bacteria are now making several substances for medical use. Vaccines too can be produced by growing harmless bacteria augmented with genes coding for the proteins (antigens) that trigger the production of protective antibodies against disease-causing microbes. Vaccines against hepatitis B, tetanus and diphtheria in humans and foot-and-mouth disease in cattle have been made using the techniques of genetic modification. Salmonella typhimurium, a bacterium commonly responsible for food poisoning, is now being used as the basis of a novel type of vaccine. Genetic engineers have removed the genes coding for certain enzymes in S.typhimurium, ensuring that it lives long enough to provoke immunity but cannot invade the body or cause disease. Genes may also be added, giving the bacterium the capacity to make antigens that trigger antibody production against many other infections, including tetanus, influenza and even malaria. Hybrid antibiotics are another prospect. These are made by altering the genes responsible for antibiotic production in fungi and bacteria, so that altered molecules are created. Hybrid antibiotics could help to circumvent the problems caused by bacteria resistant to conventional antibiotics (38).
Principles of gene therapy
Another aim of contemporary research is to combat diseases of this sort by replacing or repairing the malfunctioning gene. This would be achieved not by interfering with DNA in egg cells or spermatozoa, as with transgenic animals, but by altering genes in particular tissues of the body. Although the latter (somatic gene therapy) would help only the individual being treated, the former (germ line therapy) is not considered a legitimate target for human medicine, at least in the foreseeable future (38). Although it could prevent a serious inherited disease in future generations, therapy of this sort raises serious ethical issues while our understanding of human genetics is so limited.
The goal of somatic gene therapy is to overcome congenital diseases caused by defects in genes that normally produce particular enzymes or other proteins. One example is the thalassemia, which occurs when bone marrow cells destined to become red blood cells fail to make haemoglobin properly. Here and in similar conditions, the objective would be to reinstate formation of the missing protein by correcting or compensating for the underlying genetic deficiency.
For somatic and germ line gene therapy, three tactical approaches might be used to overcome genetic malfunctions. The first is to replace mutant genes, which fail to do their job properly, with normal ones. Conditions to be treated must have been identified and cloned, and techniques would be required for ejecting the aberrant gene and splicing in a normal version. A second alternative is to alter a malfunctioning gene to correct its erroneous message. Although this appears at least as difficult as the replacing of a faulty gene, genetic sequences have been modified in several different types of mammalian cell. The most promising approach at the moment is to introduce a fully functional gene into a cell without first removing or changing the resident, non-functional mutant gene. Whatever the technique adopted, the gene would have to be inserted into cells in the affected tissue. This is clearly a much simpler prospect for a tissue such as blood or bone marrow, which can be removed, treated in the laboratory and re-injected, than for tissues such as liver, lungs or brain.
Conclusion
Genetic improvement of animals is a continuous and complex process. Genome research is now progressing at an astonishing pace. The application of molecular biology is revolutionizing our understanding of the genetic contribution to livestock improvement. During the last ten years, molecular biology has ushered a revolution in medical genetics. Knowing the basis of a disease may lead to better therapy. There are now several large-scale collaborative research groups for different species who have undertaken genome research in the medical field.
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