Background and overview of comparative genomics. ILAR 39 (2): 048.
Comparative genomics is the cross-referencing of information on genome organization between species. The mammalian and vertebrate genomes have been highly conserved. Sequencing of the human genome is expected to be completed by the year 2003. Information from the Human Genome Project can be used to increase our knowledge of, and control over, every mammalian species of use and interest to humans, and knowledge gained from other species can be used to advance the Human Genome Project. GENES and GENOMES: Genome- full set of genes that defines an organism. Mammals and vertebrates have 2 full genomes in each cell (dipoid). The human genome contains 70,000 genes of which 10% have been identified. Proteins are made from genes to act as enzymes, transport molecules, structural proteins, hormones, or regulators of other processes. DNA is transcribed into RNA then translated into a polypeptide. A coding portion of DNA is usually about 1000bp (kb). Introns- intervening sequences of noncoding DNA; exons- coding regions of DNA (only 3% of mammalian genome, 1 per every 40-50kb). Genes are arrayed linearly along chromosomes. A karyotype is a photograph or image of chromosomes at mitosis in order of descending size and by position of the centromere. Chromosomes can be linearly differentiated by Giemsa of G-banding to produce patterns of dark and light bands along the chromosomes. G-band patterns are diagnostic for each chromosome and consistent within the species. All mammals have a genome of about 3300 million base pairs and the same number of genes (70,000). Comparative gneomics includes comparisons of every type of gene map at every level from the cytological to the molecular.
MARKERS for GENOME ANAYLSIS: Genes were originally recognized by their effect on phenotype and segregation in offspring. Genetic (linkage) mapping relies on intraspecies variation (alleles) at a particular gene locus; somatic cell genetic mapping requires interspecific variation, and physical mapping techniques require a cloned DNA probe. Molecular markers are isolated small pieces of large genomes replicated by splicing them into small self-replicating DNA molecules of bacteria or virus vectors. Lambda virus vectors, bacterial plasmids and cosmids accept 15-20 kb inserted DNA and bacterial and yeast artificial chromosomes accept foreign DNA up to a few megabases. mRNA is used to make cDNA and spliced into a vector. PCR is used to amplify a target sequence between 2 primers in a chain reaction. Base differences in DNA markers are identified as restriction fragment length polymorphisms (RFLP's) if they fall within a recognition site for a restriction enzyme. Variable numbers of tandem repeats (minisatellites) were detected by hybridizing with the core sequence and detecting different sized bands on Southern blot. A sensitive detection system to detect variation in numbers of simples sequence repeates is simple sequence length polymorphisms (microsatellites). The most useful markers for comparative genetics are the highly conserved coding genes (type II markers) which may show greater that 90% sequence homology within the exons between all mammals, even all vertebrates. Orthologues are homologous genes in different species (alpha globin in human & mouse); paralogues are genes within the same species descended from the same ancestral gene by duplication and divergence (human alpha and beta globin). One of the important criteria for gene homology is conserved map position. Most gene nomenclature is adopted from human nomenclature where homology is clear. Species designation uses a 4-letter code derived from the species name and italicized to reflect standard species nomenclature (Hsap for Homo sapiens). Additional terms worth noting are: homology- regions containing arrays of homologous genes in different species; conserved synteny- syntenic associations of the same genes in different species, regardless of gene order or interposition of other genes; conserved segment- syntenic associations of contiguous genes in different species, smallest conserved segment in different species is SCEUS (smallest conserved evolutionary unit segment); conserved order- 3 or more homologous genes in the same order in different species.
MAPPING METHODS: Genetic maps are derived from recombination frequencies between genetic markers and is related to the idea that some genes acted as if they were "linked" together in the offspring. Linkage was explained by the tendency of 2 markers on the same parental chromosome to be passed together at meiosis. The recombination percentage between 2 loci is related to how far apart they were physically. To perform genetic mapping, one must mate parents differing in 2 or more traits and then to score the patterns of segregation of parental alleles among offspring. The unit of measure to indicate 1% recombination= 1 centiMorgan (cM) (human genome 3700cM, mouse 1400cM, dog 2100cM). A physical map is constructed from information about the physical location of genes on chromosomes. This can be accomplished using radiation hybrid mapping where hybrids are constructed from donor cells that have been lethally irradiated to cause chromosome fragmentation. In situ hybridization uses a cloned probe labeled with radioactive isotope or fluorescent tag bound specifically to the DNA sequence to which it is complementary within the framework of the chromosome fixed to a microscope slide. Fluorescence in situ hybridization (FISH) is detection of the fluorescence by a sensitive ultraviolet light microscope with greater sensitivity, better efficiency and resolution but long and almost 100% homologous DNA probes. Chromosome painting is a fluorescence in situ hybridization technique that differs from FISH in that it uses a unique DNA probe derived from a whole chromosome or chromosome region. A single chromosome paint from 1 species may then be applied to chromosome preparations of another species under suppression hybridization conditions so that it binds only to homologous regions. Comparative chromosome painting (ZOO-FISH) can be performed between species reasonably closely related (humans & apes) but has poor resolution. Restriction mapping of a region of DNA is based on the recognition of specific base sequences (4-6 base palindromes) created by restriction enzymes and sequencing of the fragments.
DEPTH and BREADTH of INFORMATION: Detailed linkage maps (average distance between markers of 0.01cM) are constructed for mouse, with about 6000 genes and 13,000 DNA markers mapped. The linkage map for the rat contains 900 known genes and 4000 microsatellites. Somatic cell genetic mapping is the method of choice for rapidly (and cheaply) establishing a framework map for species like the shrew or vole in which no polymorphisms are available and breeding has been difficult. Comparative gene mapping has shown a level of conservation with large regions of conserved synteny with the human genome. There is a level of conserved synteny between birds, fish and humans. Comparative chromosome painting has confirmed the conclusions of comparative mapping- that the genome has been very conserved at least between eutherian offers that diverged about 60 MYA.
PRACTICAL VALUE OF COMPARATIVE GENOMICS: Since the mammalian genome is very conserved, it makes sense to combine the gene mapping information from humans and nonhuman animals for the mutual benefit of both. The location of disease genes in humans can immediately assist in identification of the homologous condition in animals. Treatments and cures can also be used to treat the homologous human or animal disease. A detailed gene map of an animal species can assist animal breeding by selecting for breeding out a trait. A gene responsible for a disease or a superior trait is isolated, sequenced and translated into the amino acid sequence of its protein product. Quantitative trait loci (QTL) are economically important traits each with a relatively small effect (meat quality, wool fiber, fertility) and has lead to the branch of genetic analysis known as quantitative genetics. Nonhuman mammals are advantageous for genetic study because of their shorter generation time, large family size, ability to set up crosses, target disruption of a gene "knockout", or insert a foreign gene (transgenic).
DEDUCING the ANCESTRAL MAMMALIAN GENOME: Look at karyotypes to determine the relatedness of the species to one another. Karyotype arrangements shared by 2 species either could be ancient and retained by both or could be the result of a recent change within that particular lineage.
CONCLUSION: Comparative genomics has allowed the genetic and physical maps for a variety of species using a variety to methods. Comparative mapping enables transfer of information between human, livestock and rodent genomes. Comparisons of location can be used to identify homologous genes involved in disease states in humans, domestic animals, and rodent models, making possible the development of techniques for diagnosis and treatment for transfer in either direction. Comparative gene mapping should greatly speed up the search for genes that specify inherited diseases in mammals and humans, as well as genes that specify economically important traits.
> Matching for 1-4: Molecular markers, 1 centiMorgan (cM), Flourescence in situ hybridization (FISH), Chromosome painting
1. __________________are isolated small pieces of large genomes replicated by splicing them into small self-replicating DNA molecules of bacteria or virus vectors.
2. The unit of measure to indicate 1% recombination= _____________________
3. ______________________is detection of the fluorescence by a sensitive ultraviolet light microscope with greater sensitivity, better efficiency and resolution but long and almost 100% homologous DNA probes.
4. ______________________is a fluorescence in situ hybridization technique that differs from FISH in that it uses a unique DNA probe derived from a whole chromosome or chromosome region.
5. Comparative chromosome painting or ___________________ can be performed between species reasonably closely related (humans & apes) but has poor resolution.
1. Molecular markers
2. 1 centiMorgan (cM)
3. Flourescence in situ hybridization (FISH)
4. Chromosome painting
Internet comparative mapping resources. ILAR 39 (2): 066.
This article briefly describes internet databases and resources providing comparative genomic information. Internet-based resources have become a vital tool for the comparative mapper. Genome databases have been established on an organism-specific basis, usually with a proprietary database structure and delivery system. The largest database is the Human Genome Organization's database at Oak Ridge National Laboratory. The most comprehensive comparative database is the Mouse Genome Database at The Jackson Laboratory. This lists mammalian homologies and includes a powerful set of search criteria.
The basic type of comparative linkage comprises shared symbols and names and includes the use of symbols from other species as an alias. This system is inflexible with regard to incorporating deeper understanding of gene homologies and to nomenclature changes. Newer databases are including development of automated software, which will explore existing databases and create their own database of links between different species databases based on a defined set of homology criteria.
Comparative mapping using chromosome sorting and painting. ILAR 39 (2): 068.
Mammalian species have been used to develop comparative gene mapping at the cell level. These maps are constructed by whole chromosome-specific, fluorescence-labeled DNA probes, by random-primed amplification of flow-sorted chromosomes.
Gray (et.al., 1975) used the technique of flow cytometry to sort chromosomes and measure their DNA content. Chromosomes can be separated by size and sorted into their respective types. These techniques were used to prepare chromosome-specific libraries for constructing chromosome maps.
Chromosome-specific libraries made from flow sorting were used in FISH (fluorescence in situ hybridization) experiments to paint chromosomes and analyze chromosome rearrangements. These FISH preparations were examined using digital fluorescence microscopy and image processing.
Meltzer (et. al 1992) made chromosome specific probes from microdissected chromosomes or parts of microdissected chromosomes. This technique is used in particular for comparative painting with avian and other species which have multiple microchromosomes and are consequently difficult to sort.
Fluorescence measurements from each chromosome accumulate in the FAC (=fluorescence-activated cell sorter) computer and are used to construct a flow karyotype. Flow karyotypes are made of clusters of signals which represent one or more chromosome types arranged according to size and base-pair ratio.
Flow karyotypes are distinct for each individual and each species. Typically, most closely related species tend to have similar flow karyotypes. Variation within a species is due to different amounts of noncoding repetitive DNA, present mostly on centromeric heterochromatin. The flow karyotypes of different inbred strains of the same species (e.g. mice) can be distinguished because they are homozygous for different centromeric heteromorphisms.
CROSS-SPECIES CHROMOSOME PAINTING
The human and the mouse are two mammalian species with the most comprehensive genetic maps. The mouse genome has been extensively rearranged during evolution, compared with the human genome. Cross-species comparative painting can be relied on to indicate regions of genetic map homology.
Studies indicate that cross-species painting is more reliable than conventional cytogenetics using Giemsa banding in determining chromosomal homlogy between species. Primates studied currently have a number of chromosomes that are painted exclusively by 1 human chromosome-specific painting probe. Rearrangements that have occurred during the evolution of humans and great apes have been revealed by chromosome painting with human probes. Specifically, homologues of human chromosomes 14 and 15 are separate in the great apes but linked in lower primates and nonprimate species. Other types of association help to link other species within a particular branch of the phylogenetic tree.
KARYOTYPE EVOLUTION IN MUNTJAC SPECIES
Reciprocal cross-species chromosome painting has been achieved and published in many species, including muntjacs. Chiness and Indian muntjacs have 46 and 6-7 chromosomes, respectively. However, the phenotypes of these species are similar, and they breed to produce viable, but sterile offspring. The Indian muntjac has large chromosomes, but the lowest chromosome numbers of all mammals. Each of this species chromosomes can be separately painted in one of three colors.
Chromosome-specific painting probes have been generated by DOP-PCR amplification of sorted chromosomes from many species. Across all mammals studied, relatively large blocks of chromosomal DNA have been transmitted virtually unchanged throughout evolution. The Indian muntjac is an example of how easy cross species comparisions of the genomes of mammals can be made to guide the genetic mapping of unmapped species. Additionally, cross-species chromosome painting has great potential in determining phylogenetic relationships of other mammalian species, as well as being a great tool to understand the nature of chromosomal reorganization in evolution.
1. T/F In general, more closely related species show fewer rearrangements than more
distantly related species.
2. Define the following abbreviations: PCR; FISH; FACS; and DOP.
3. Fill in the missing word regarding flow karyotypes:
Variation within a species is due largely to the presence of different amounts of noncoding
repetitive DNA, present mostly as ____________ heterochromatin.
4. Do most closely related species tend to have similar or different karyotypes?
5. If a species has low chromosome numbers, what does this indicate about the species
chromosome size and base-pair ratio?
6. Name the two mammalian species with the most comprehensive genetic maps.
7. Which species has the lowest chromosome number in mammals:
a. brown brocket deer b. Chinese muntjac
c. Indian muntjac d. none of the above
2. PCR = polymerase chain reaction;
FISH = fluorescence in situ hybridization
FACS = fluorescence-activated cell sorter
DOP = degenerate oligonucleotide-primed
5. larger chromosome size and show less variation in base-pair ratio
6. human and mouse
Comparative painting of primate genomes. ILAR 39 (2): 077.
SUMMARY: Gene mapping of multiple species has been underway since the 1970's. In spite of this, only the mouse has been extensively mapped, while other species have lagged significantly behind. Mapping become more rapid with the introduction of fluorescence in situ hybridization (FISH) using human specific DNA probes to map nonhuman chromosomes. At this time, many probes are available. One of the most attractive probes are the chromosome paints (labeled mixtures of DNA sequences derived from flow-sorted or microdissected chromosomes that are specific for a single chromosome and hybridize to the entire chromosome in a target metaphase).
Within primates, approximately 20 species have been analyzed by chromosome painting. This article summarizes these results and provides some hypothesis regarding the origin and evolution of the primate genome.
Although chromosome morphology was traditionally used to study the evolutionary processes of genomic changes, recent studies with chromosome painting in high rearranged genomes such as gibbons show that cytogenic phylogenies must be based on homologies established on the basis of DNA content.
Chromosome rearrangements are believed to be rare, so identical rearrangements in different phylogenetic lines are believed to indicate a common evolutionary origin.
Common chimp (Pan troglodytes), lowland gorilla (Gorilla gorilla), and Sumatran subspecies of the orangutan (Pongo pygmaeus abelii) have been analyzed using human chromosome paints. It was found that human chromosome 2 hybridized to 2 pairs of homologous chromosomes in all great apes, suggesting these homologues are ancestral. Recent research has found that the proposed ancestral fusion point leading to modern human chromosome 2 is close to band 2q12. Interestingly, molecular cytogenetic data for human chromosome 2 and its great ape homologues show that it cannot be used to phyogenetically link human and chimpanzees to the exclusion of the gorilla.
Chromosome painting has also shown that there was a reciprocal translocation in the gorilla between homologues to human chromosomes 5 and 17.
Hylobatids have traditionally been classified with the great apes and humans in the superfamily Hominoidea. However, chromosomal analysis showed that there was not karyological relationship with the great apes. Hylobatids have highly rearranged genomes. Only the X chromosome and the analog to human chromosome 21 have not been involved in translocations. The hylobatids also exhibit extensive chromosome polymorphisms including inversions and translocations. There is evidence that the translocation has survived speciation events. This supports the proposition that lesser apes are more appropriately classified with the monkeys instead of the great apes and humans. It is unclear if the extensive chromosomal polymorphisms are due to a higher chromosomal mutation rate or if the mutations are just more easily fixed in this population (monogamous catarrhine species with nuclear family units and arboreal lifestyle).
Old World Monkeys: Two families, Cercopithecidae and Colobidae. Tend to be chromosomally conservative (especially as compared to lesser apes).
Include Papio, Macaca, and Cercocebus. Human chromosome syntenies, except for chromosome 2, are found in the macaque karotype. Three macaque chromosomes painted with human specific paints and indicated chromosome fusion/fission events and species differences limited to few inversions. Karyotype is 2n=42.
Nearly all have 2n=44. Only difference between African and Asian colobines was presence of small pair of acrocentric chromosomes in Asian species. Some Asian colobines have more derived karotypes than African colobines, macaques, great apes, and humans.
New World Monkeys: Much more karyologically variable than expected.
Relatively uniform cytogentically. 2n=44 to 48. Callithrix jacchus has the only cytogenetic data with disturbed syntney of 7 human chromosomes and 9 systenic associations (multiple hybridization signals on single chromosomes) not found in humans.
Capuchins have multiple hybridization signals on different chromosomes for 8 human paints and 6 associations exist that are not found in humans.
Spider monkeys exhibit dispersed signals over the karyotype and have 25 chromosomal associations not found in humans.
Howler monkeys reveal fragmentation and translocation of most human chromosomes. There is an exceptionally large number of chromosome rearrangements separating the karotypes of at least three species of howler monkeys, too.
The associations found in the New World monkeys can give information regarding the ancestral conditions and divergence patterns. However, additional study is needed to adequately determine these patterns.
Analysis shows that lemurs have undergone only a few reciprocal translocations with other translocations that make them significantly different from other primates. This urges scientists to use caution when calling the lemur karyotype the closest to the ancestral version their translocations may have moved them significantly far from that designation.
The loris are presumed to maintain a more primitive karyotype but there is not really any information on chromosome painting in the literature.
We are starting to get a better understanding of the cytogentic relationship and how it relates to the phylogenetic relationships of the primates. However, additional study is needed to fully understand the relationships between the various species throughout the primates.
1) What are the scientific names of the (a) common chimpanzee, (b) lowland gorilla, (c) orangutan, and (d) pygmy chimp?
2) Who are the hylobatids?
3) Old World Monkeys are the ___________
4) New World Monkeys are the __________
5) Capuchin monkey scientific name is ___________
6) Black-handed spider monkey scientific name is __________
7) Howler monkey genus is ______________
1) (a) Pan troglodytes, (b) Gorilla gorilla, (c) Pongo pygmaeus, and (d) Pan paniscuc
5) Cebus capucinus
6) Ateles geoffroyi
Chromosome painting in marsupials. ILAR 39 (2): 092.
To generate a model of mammalian karyotype evolution and to extend our knowledge of the evolutionary relationships of mammals, it is helpful to deduce ancestral chromosomal arrangements. The genomic arrangement in a common ancestor could be deduced by comparing the ancestral genomes of the 3 groups deduced from interspecies and interordinal comparisons. However, it has been difficult to construct an ancestral mammalian chromosomal arrangement because of the wide variety of karyotypes observed in mammals. Neither comparative chromosome banding nor chromosome banding studies are adequate for this tasks. Comparative chromosome banding has provided a powerful tool to establish interspecies chromosomal homologies within some orders. Chromosome bands lack the detail necessary to establish homology except over large regions and are reliable only between closely related species. Comparative mapping of many individual genes in several species provides a good test of genetic homology, which has revealed the presence of large chromosomal segments conserved in the vertebrate genome for up to 400 million yr. However, it is time consuming, and whole chromosomal homology must be inferred from limited single-gene comparisons.
A new strategy for analyzing chromosomal evolution and for comparing genomes in mammals is comparative chromosomal painting, performed by fluorescence in situ hybridization (FISH) using probes derived from flow-sorted or microdissected whole chromosomes or microdissected chromosome arms or segments. Chromosome painting provides information on chromosomal homology, chromosomal rearrangements, and syntenic groups of genes; this information greatly advances our understanding of genome evolution. Comparative chromosome painting with human-derived DNA probes has been demonstrated in great apes, old world monkeys, and in prosimian lemurs. It may also be used to demonstrate chromosomal homologies between more distantly related eutherians. Human paints have been hybridized to the chromosomes of nonprimate mammalian species such as horse, pig, muntjac, cat, and even whale and mouse to elucidate chromosomal rearrangements in relation to human . This has allowed us to establish chromosomal homologies across the Eutherian and should make it possible to establish an ancestral eutherian karyotype.
Marsupials diverged from eutherian ("placental") mammals about 130 million yr ago and monotremes about 170 million yr ago. Marsupials have low diploid numbers and exceptionally large and well-differentiated chromosomes. They are well studied by classic cytogenetic techniques. Their karyotypes show a startling degree of homology even between distantly related groups. Because of the limited number of chromosomes in the tammar wallaby, flow-sorting and singly sorting the chromosomes and microdissecting the chromosomes were possible.
Swamp wallaby has a highly derived karyotype with the low chromosome number (2n=10 male; 11 female). Comparative painting with tammar wallaby probes showed that there has been minimal genomic shuffling, and the unusually low diploid number in swamp wallaby is the result of tandem fusions. Dasyurids diverged from macropodids about 50 million yr ago. The successful hybridization of tammar paints onto the distantly related dasyurid chromosomes ensures that we can use the technique to contradict or confirm that hypothesis of the ancestral 2n=14 karyotype.
The mouse gene map. ILAR 39 (2): 096.
Origin and History of the Mouse Map The first mammalian autosomal linkage group was identified in the mouse. The dense map of the mouse, the high degree of linkage conservation between mouse and human genomes, and the interest in comparative mapping between the mouse and human genomes have enhanced the value of the mouse as an experimental model for human disorders. William Ernest Castle greatly influenced early mouse genetics. He presented some of the first findings relative to the segregation and independent assortment of various mouse coat colors. Clarence Cook Little created the first inbred strain in 1909 to study cancer genetics and resistance to transplanted tumors. This first inbred strain was DBA, a line of dilute (d), brown (b), nonagouti (a) mice. The mouse genetic map was begun in 1915 when Haldane and others published a landmark paper linking the pink-eyed dilution and albino traits in the mouse. Mapping methods in the mouse up through the early 1970s were primarily dependent on observation of the segregation of phenotypic traits in linkage crossings using inbred strains. The advent of chromosomal banding permitted the identification of all mouse chromosomes, and the combination of banded chromosomes and reciprocal translocations was used to assign linkage groups to most chromosomes. Linkage groups could finally be assigned to all chromosomes of the mouse genome by 1976. In 1972, an official chromosomal numbering system was first established by the Committee on Standardized Genetic Nomenclature of the Mouse to eliminate conflicting terminology in the literature. Margaret C. Green exerted the main effort in synthesizing mapping data and maintaining the mouse map for more than 20 years (1950s to 1977).
Advances Contributing to the Map
During the 1970s-80s, several significant advances dramatically increased the flow of mapping information. These advances included the discovery of additional polymorphic loci, recombinant DNA technology, selection of the mouse as a model organism in the Human Genome Initiative, development of interspecific backcrosses, and creation of genetic databases as communication tools. Mus spretus (interspecific crosses) and Mus musculus castaneus (intersubspecific crosses) are polymorphically different from laboratory mice. Both have been used in linkage studies. However, hybrid F1 males between laboratory mice and M. spretus mice are sterile, precluding the more efficient intercrosses for high-resolution mapping more commonly done with M. m. castaneus. The discovery and development of DNA polymorphisms made it possible to store DNA from crosses and cumulatively type the DNA panels for additional markers as they were identified. Use of restriction fragment length polymorphisms (RFLPs) was based on naturally occurring differences in enzymatic cleavage patterns in the genomes of divergent strains. PCR amplification of highly polymorphic loci (i.e. microsatellites, simple sequence length polymorphisms or SSLPs) permitted the construction of a scaffold spanning the length of the mouse genome on which to build a descriptive map. Physical maps of the mouse genome have been slower to develop compared with the human genome.
Methods used to order and assign loci in mouse chromosomes are genetic linkage crosses, recombinant inbred (RI) strains, recombinant congenic (RC) strains, congenic strains, somatic cell hybrids, and in situ hybridization of probes to chromosomes. Future mapping will include increased use of radiation hybrids (RH) and physical mapping using overlapping DNA segments. Genetic crosses still contribute to the genetic order of loci in mapping. Haplotype analyses of progeny from backcrosses and intercrosses enable estimation of genetic recombination for alleles at 2 or more mutant genes or polymorphic loci segregating in a cross. Genetic recombination is the result of physical crossing over between homologous chromosomes. Genetic distance and ordering of loci are derived from the number of crossover events between loci divided by the total number of progeny genotyped. Interspecific crosses with M. spretus generate only female maps because of the sterility of F1 males. For most parts of the genome, genetic recombination distances are greater in female mice than in males. RI strains are sets of inbred strains created from sibmated F2 progeny produced by crossing mice from different inbred progenitor stains. Cross-over events can be detected by strain distribution patterns (SDPs) of alleles among the RI lines, typically using a series of regional markers (i.e. biochemical markers, RFLPs, PCR gene analysis, microsatellite polymorphisms). RI strains are valuable for mapping phenotypic or quantitative traits that differ between the progenitor strains. RC strains are derived in a manner similar to RI sets except 1 or more backcrosses to 1 parental (background) strain are made after the F1 generation before inbreeding is begun. The other parental strain is designated the donor. The number of backcrosses preceding inbreeding determines the proportion of background and donor genomes. As with RI strains, a detailed characterization of SDPs of genes within a strain set is used to determine linkage relationships between loci and chromosomal segments associated with a trait. Congenic strains are derived by successive backcrosses in which 1 strain (donor) donates a segment of chromosome to the recipient (background or host) strain. Somatic cell hybrids are derived from the fusion of cells from unrelated species to generate cell lines containing a subset of mouse chromosomes in an otherwise foreign host genome. Somatic cell hybrids enable the quick mapping to chromosomes of genes for which no polymorphic differences have as yet been identified. Fluorescence in situ hybridization (FISH) with multiple probes labeled with different fluorochromes allows the physical ordering of genes on a chromosome. Complementation analysis using a series of overlapping deletion mutations allows loci within the deletion region to be unambiguously ordered and has proved valuable in the identification of new recessive mutations in chromosomal regions and subregions. RH mapping can potentially provide high-resolution gene ordering for nonpolymorphic regions and fine mapping of regions not separable in the recombination map. Because each RH cell line represents a highly fragmented subset of the mouse genome, error detection is difficult and a framework map tied to the well-defined genetic map is needed.
Current Map Status
The mouse genome is estimated to contain approximately 300,000 megabases of DNA. Genes represent discrete expressed elements of the genome defined molecularly or by the segregation of a single phenotypic trait. Pseudogenes have coding sequences but lack introns and are not expressed. Quantitative trait loci (QTL) represent regions of the genome associated with particular phenotypes displaying complex inheritance. DNA segments are anonymous loci recognized by variation in DNA sequence. Table 1 (p. 102) summarizes what is known about the types of genes by chromosome as of September 1998. Graphical maps depicting gene arrangements on mouse chromosomes come in several primary forms: cytogenetic, linkage, physical, and comparative. Cytogenetic maps depict the approximate location of loci relative to chromosomal bands identified by karyotypic methods. Up through the mid-1980s, cytogenetic mapping was done using genetic crosses in which chromosome anomalies were segregating. Currently, such mapping is done with in situ and fluorescent in situ (FISH) hybridization of molecular probes to banded chromosomes. Linkage maps represent the relative positions of loci on a given chromosome. Genetic distances are calculated in centi-Morgan units from recombinational frequencies between genes derived from mouse crosses. Physical maps display an ordered array of genes, anonymous probes, and STSs relative to a set of overlapping clones (i.e. BACs, YACs, cosmids). Comparative maps illustrate homologous associations among species. These maps reveal regions of evolutionarily conserved chromosomal segments between species.
Comparative genomic research using the mouse falls into 2 broad categories. The first includes studies that attempt to gain evolutionary insights into genome organization. The second includes studies designed to utilize a well-defined genetic map in 1 species to accelerate mapping of the genome of a poorly mapped species or to identify and clone candidate genes for human genetic diseases. One concern associated with comparative genomic research is whether true orthologues are being observed in the species being considered. Orthology describes genes in different species that derive from a common ancestor. Orthologous genes may or may not have the same function. Paralogy describes homologous genes within a single species that diverged by gene duplication. High sequence similarity can occur among both orthologous and paralogous genes, but low sequence similarity is also possible between orthologous genes. The recommendation of the Human Genome Organization Committee on Comparative Genome Mapping is that orthology be determined by sequence similarity in conjunction with a conserved map position between homologous markers. The ability to map and sequence genes has outpaced the ability to attribute functions to them. Expressed sequence tags (ESTs) serve as probes that allow the specific identification of nucleotide sequences that are likely to be expressed. The use of ESTs as routine gene mapping tools has resulted in the generation of "transcript maps."
Comparative Mapping Identifies Human Genes Underlying Complex Traits
Contiguous gene syndromes are conditions that result from deletion or duplication of whole chromosomal segments with many genes. The resulting abnormalities are presumed to be a mixture of single gene effects and, for duplications, gene interactions. Dissecting out single gene effects is virtually impossible due to the complex nature of such multigenic conditions. An example of such a syndrome is trisomy 21 that causes Down syndrome, one of the most common causes of mental retardation. The Ts(1716)65Dn (abbreviated Ts65Dn) mouse is a model for this syndrome that was created based on detailed comparative mapping. These mice show developmental delay early in life and severe behavioral and learning deficits. Many human diseases (i.e. diabetes, some forms of cancer) have a genetic component that cannot be associated with a single mutant gene. The genes underlying such diseases often can be identified by making specially constructed mouse stocks that isolate individual genes implicated in similar mouse diseases. Comparative mapping and sequence comparison has also been used to identify genes underlying mammalian circadian behavior.
Future Development of the Map
New types of maps will appear as "functional genomics" begins to develop. Not only is there interest in where genes are located on chromosomes and how they are organized (physical map), but also where and when they are expressed, how they are regulated, and how they interact. Transcript maps, expression pattern maps, and maps describing metabolic and developmental cascades will be created to graphically represent genome function. Synthesis of genetic, physiological, expression, and systematic information will facilitate construction of the "complete" mouse map and its functional blueprint. The realization of the "complete" mouse map is only a matter of time, and beyond the map is ultimately our understanding of the biological process. Table 2 (p. 129) lists some of the currently available database resources for mouse-related genome data. For those of you feeling particularly masochistic, the mouse linkage map as of September 1998 is shown on pp. 104-126 on a chromosome by chromosome basis.
1. Order, family, subfamily, genus and species for mice? What is the diploid number for mice?
2. What color would a mouse with the following genotype be? Why? AaBBccdd
3. What do the following acronyms stand for? YAC, BAC, PAC, RFLP, SSLP, FISH
4. Which of the following Mus sp. is used for high-resolution mapping of mutations via intercrosses with Mus musculus?
A. Mus musculus musculus
B. Mus spretus
C. Mus musculus castaneus
D. Mus spicilegus
E. Mus musculus domesticus
1. Rodentia, Muridae, Murinae, Mus musculus. 2N=40
2. This animal [AaBBccdd] would be white or albino because the C gene codes for pigmentation. CC and Cc are pigmented but the homozygous recessive cc is white no matter what alleles the animal has for the other coat color genes. This phenomenon is referred to as epistasis the interaction of nonallelic genes in which an allele of one gene masks the phenotypic expression of the allelic alternatives of another gene or genes. Epistasis is not dominance. The C gene is epistatic to the A, B, and D genes whereas the A, B, and D genes are hypostatic to the C gene.
3. YAC = yeast artificial chromosome; BAC = bacterial artificial chromosome; PAC = P1 artificial chromosome (bacteriophage P1); RFLP = restriction fragment length polymorphism; SSLP = simple sequence length polymorphism; FISH = fluorescent in situ hybridization.
4. C. Mus musculus castaneus
The rat gene map. ILAR 39 (2): 132.
Until the 1980s 2 main drawbacks with the rat as a genetic model organism have been the poor development of the rat gene map and the paucity of useful polymorphic genetic markers. The most important advance has been the discovery and development of microsatellite markers and new regional chromosome mapping methods such as fluorescence in situ hybridization. In the early 1970s, the rat banded karyotype was characterized and standardized. The major rat genome database is RATMAP and reports are published in the journal Rat Genome. The homology Database at the Mouse Genome Database works with RATMAP to compile updates on rat-mouse and rat-human homologies. Markers that have been mapped in the rat by be subdivided into 2 major groups: (1) those corresponding to genes (type I markers), and (2) anonymous DNA markers (mostly SSLP markers, type II markers). Currently, mapping information exists for about 900 type I and 4000 type II markers. About 100 lice have been mapped regionally both by FISH and by linkage and these can be used to "anchor" the linkage groups on the physical chromosomes. Based on the status of the maps at this time, it is possible to distinguish 70 autosomal chromosome segments, each containing 2 or more genes that appear to have been conserved between rat and human. The corresponding comparison between rat and mouse yields much longer (and therefore fewer) conserved segments, about 33 (and perhaps as many as 61). What appears to be a considerable amount of genome rearrangement between rat and mouse is perhaps more than would be expected on the basis of evolutionary distance. Since the rat and mouse shared a common ancestor about 25 million years ago, the rate of chromosomal evolution in rodents is comparatively rapid among mammals.
1. What is SSLP an acronym for?
2. What is FISH an acronym for?
1. Simple sequence repeat length polymorphism
2. Fluorescence in situ hybridization
The vole gene map. ILAR 39 (2): 138.
Gray voles belong to the family Arvicolidae and the superfamily Muroidea. In this article the authors describe results of mapping the genome of five species of gray voles: M. arvalis, M. rossiaemeridionalis, M. kirgisorum, M. transcaspicus and M. agretis. M. agretis is widely spread throughout Europe and Asia and is often used in laboratory studies since its X chromosome, which has the largest block of heterochromatin, is very easy to identify. Voles are used as an experimental model for evolutionary, phylogenetic, and genetic studies. Mapping genomes is extremely valuable not only for developing genetics of any particular species but also for conducting comparative studies of chromosome evolution in different mammals. In this study by mapping the X chromosome of the various vole species, this data could be useful in reconstructing the ancestral mammalian X chromosome. Authors used several different approaches to localize X-linked genes. They first used an analysis of gene expression in reciprocal hybrids, among four species. The second method they used was somatic cell hybridization. These two methods did not reveal the position of the genes on the chromosome, therefore the third method authors are currently working on is to create a cytogenetic map.
Gene maps of nonhuman primates. ILAR 39 (2): 145.
A. Reasons for mapping these species
a. Primates are important as animal models of human disease: Development & use of detailed gene maps will allow opportunities for animal studies that identify new genes affecting individual variation in people in their risk to develop a certain disease.
b. Linkage mapping studies provide opportunities to localize and identify gnes that influence normal physiological, anatomical and behavioral variation.
c. Can help researchers in recognizing homology between NHP genes and human genes.
d. Investigate the evolution of chromosomes, genes and complete genomes: this allows the direct analysis of changes through time in gene complement and chromosome organization in primate evolution.
B. Current status of the maps
Few NHP species and loci have been analyzed thoroughly because the research has been done by investigators with different research perspectives and goals. Somatic cell hybridization was originally used, but is being replaced by fluorescence in situ hybridization (FISH).
NHP gene mapping in most species has been diffiuclt because of a lack of resources. Genetic linkage mapping has been limited because there are few multigeneration pedigrees available.
C. Approaches used to develop the maps
FISH is now the most common method used. It can map single-copy DNA segments to particular regions in the NHP chromosomes. This method is better than somatic cell hybridization because it allows the sublocali-zation of a gene or DNA segment to a smaller region in a chromosome. FISH doesn't require polymorphism at the locus being mapped.
Genetic linkage mapping(a.k.a. recombinant or meiotic mapping) has been used in a small number of studies. This method requires that the gnen or DNA segment to be mapped exhibit intraspecific polymorphism and the analysis of genetic variability among several hundred pedigreed animals. It can establish the order among tightly linked loci and make it possible to locate new genes that influence phenotypic traits. This method can be used to investigate the genetic variation related to human diseases or genes that influence normal variation in anatomy or physiology.
Radiation hybrid mapping has not yet been used extensively in NHPs. To construct a radiation hybrid panel for human gene mapping, human fibroblasts are irradiated at a dose lethal to the cells, which induces frequent breaks in all the chromosomes. These fibroblasts are fused with a series of rodent cell lines and grown in culture. To map specific DNA segments or genes, each part of the panel of radiation hybrid cell lines is scored for the presence or absence of the DNA segment of interest. A radiation hybrid panel is available for babbons. This method is not dependent on known polymorphisms in the DNA segment to be mapped.
D. Scientific contributions of the maps
The major contribution has been an improved understanding of the evolutionary histor of NHP genome orgaization. Direct comparisons with the human genome have provided insight into the origin of human chromosome sturcture. Example: inversion on chromosome 18 and a fusion that produced human chromosome 2 are unique to man.
E. Anticipated future contributions of the maps
It will lead to more detailed description of genomic differences among primate species, including man. Linkage maps will help to localize and identify genes that influence risk factors for disease and affect normal variation.
The cattle gene map. ILAR 39 (2): 153.
Cattle are perhaps one of the oldest domesticated species of animals, and it is currently thought that at least 2 different domestications are represented in current breeds of cattle. Bos primigenius primigenius is thought to be the ancestor to the humpless breeds of cattle (Bos taurus), while Bos primigenius namadicus is thought to have given rise to the humped zebu breeds (Bos indicus). Both of the current species are interfertile, have 29 autosomes and a submetacentric X chromosome. The morphology of the Y chromosome is the only recognized structural difference between B. taurus and B. indicus karyotypes. Cattle are members of the subfamily Bovinae, the family Bovidae, suborder Ruminatia and order Artiodactyla. Gene nomenclature follows the guidelines for human gene nomenclature as recommended by the International Society of Animal Genetics. Loci without human equivalents are named according to the recommendations of the Committee on Genetic Nomenclature of Sheep and Goats.
Through comparative mapping, it appears that cattle and cats have genomes more highly conserved relative to the human genome than the mouse genome. Gene mapping in cattle have concentrated on the potential for marker-assisted selection (MAS) of desirable and marketable traits. Genetic markers of advantageous alleles for economic trait loci (ETL) and guantitative trait loci (QTL) have been identified. ETL and QTL can be mapped by linkage analysis. Markers mapped in proximity to ETL can be used to assist in selection of the ETL if the recombination frequency is sufficiently small and the chromosomal phase of marker and ETL alleles is known. Efficiency of MAS can be increased by identifying markers on either side of the ETL.
Another goal of gene mapping is to identify and clone genes responsible for ETL. Map based cloning (reverse genetics or positional cloning) is the process whereby the application of map information is used to clone a gene responsible for a specific trait in the absence of information about the biochemical or molecular basis of the trait. Cloning genes for ETL in livestock, however is almost prohibitive, since naturally occurring chromosomal deletions of important genes, important tools in many of the human and mouse successes, have not been identified and propagated in livestock.
As of the date of publication of this paper (1998) 400 type I loci had been mapped in cattle, primarily through somatic cell genetics. In situ hybridization, especially with fluorescence (FISH), has been used effectively to address the order of type I loci, to assign syntanic groups to specific chromosomes and to anchor the rapidly growing linkage map to chromosomes. Using the FISH method, the linkage map had been anchored at approximately 100 sites on all the bovine chromosomes. Of the 400 loci that had been mapped in cattle, most had also been mapped in mice and humans. Conservation of synteny (genes known to reside on the same chromosome in different species) has been observed between cattle and humans, however conservation of linkage (gene order) may not be as prevalent and researchers have demonstrated rearrangement of linear order of genes in BTA 7/HSA5. Somatic cell genetics is a common method for building synteny maps. Hybrid somatic cells can be constructed so that the chromosomes of practically any progenitor species are preferentially lost. Since chromosome loss is more or less random, each clone will retain a different subset of chromosomes from the species being mapped. Concordance of retention is evidence for the location of 2 genes on the same chromosome. Discordance of retention is evidence for asynteny(location on different chromosomes).
Unique DNA sequences, repetitive elements, and whole genomes have all been effectively localized to chromosomal sites by in situ hypridization. This technique employs the attachment of a microscopically detectable marker to a DNA probe followed by hybridization of the probe to denatured DNA of an otherwise intact chromosome. FISH provides superior spatial resolution, usually requiring visualization of fewer labeled chromosomes; it is faster; and the probe employed is generally more stable. Use of Mutiple probes with different color signal on the same chromosomes provides the potential for ordering loci within the resolution limits of approximately 100 kb. Linkage maps are defined in meiotic rather than physical terms, with a map unit representing 1% recombination. Linkage mapping requires detection of maternal and paternal alleles in gametes produced by heterozygous individuals; both polymorphism and large number of offspring are requirements for linkage mapping.
A growning number of traits of economic significance are being placed on the bovine genome map. Bovine leukocyte adhesion deficiency (BLAD), and uridine monophosphate shnthetase deficiency (UMPS) have been mapped to specific sites on chromosome 1. BoLA was shown to be associated with susceptibility to leukemia virus infection. The polled locus was linked to microsatellites on chromosome 1 and Weaver disease maps to markers on chromosome 4. This is also associated with a quantitative trait for improved milk production. Variation around the prolactin gene on chromosome 23 is related to milk production in some holstein sire families. Microsatellite mapping ahs located an additional 5 QTL for milk production. The Muscular hypertrophy (double muscling ) trait was mapped to microsatellite markers on chromosome 2 and comparative candidate positional cloning suggested myostatin as a candidate gene. BLAD in holstein cattle has been identified as a missense mutation coding amino acid 128 in CD18. Once identified, it was possible to distinguish the mutant and normal allelles by PCR, providing the ideal genetic marker of this economic trait locus and allowing producers to identify and eliminate that trait from their breeding herds
In a brief search of the literature since 1998, two additional papers describing QTL scans for economically important traits in US holstein cattle, and carcass and growth rates important to beef producers have been published.
The sheep gene map. ILAR 39 (2): 160.
Most, if not all, gene mapping work has been done on the various breeds of domestic sheep (Ovis aries).
Reasons for mapping this species include- enhance the quality and diversity of sheep and their products, by positive or negative marker selection, improve animal health and welfare by marker assisted selection to eliminate diseases and to protect the environment by reducing the need for widespread use of agrochemical, investigate speciation and evolution, establish models for the human genetic disease, and to protect genetic diversity.
Current map status-
627 loci, 245 known genes have been mapped- 2 linkage maps and 1 cytogenetic map.
Extensive use of cattle micosatellite markers, which facilities comparison between ovine and bovine maps, the linkage groups show extensive conservation.
Reference Flocks- 1)IMF, comprising 9 pedigrees each of 3 generations 2) CSIRO mapping flock of 15 3 generation full sib families. 3) USDA Meat Animal research center Clay center flock.
Resource flocks- disease resistant flocks
Parasite resistance-Romney and Coopsworth sheep selected for resistance and susceptibility to internal parasites.
Facial eczema-(mycotoxicosis caused by the ingestion of the hepatatoxin sporedesmin produced by the fungus Pithomyces chartarum)
Romney sheep selected for resistance and susceptibility . Rye grass staggers (toxin lolitrem B of an endophtyic fungus in rye grass)
The horse gene map. ILAR 39 (2): 171.
1. The first contribution to the horse gene map was identification of linkage between 6-phospho gluconate dehydrogenase and the K blood group system.
2. Gene mapping discoveries were incidental to studies of new genetic systems for parentage analyses in horses.
3. Genomics: area of research involved in identifying, sequencing and mapping large numbers of genes.
4. Evolution of Equus is an example of evolution associated with rapid karyotypic change. Member species are clearly delineated by differences in chromosome numbers: range from 32 in Hartmann's zebra to 66 for Prezwalski's horse.
5. Equine species can be induced to hybridize, but most hybrids are sterile. Most common hybrid is mule.
6. Chromosomes are identified based on chromosome banding patterns produced after chemical treatment.
7. Comparative gene mapping: area of research that uses data from human genome studies to predict the genomic organization of another species without actually sequencing that species' genome.
8. Linkage map allows identification of DNA markers that are closely linked to genes for inherited diseases, performance traits or other inherited characteristics, in this case for the horse.
9. First comprehensive genome map for the horse was a comparative map. Developed using ZOO-FISH(chromosome painting) it identify regions of homology between man and horse genome.
1. How is the mule produced?
2. What is the chromosome number for the following species?
1. Cross between a male donkey(Jack) and female horse(mare).
The dog gene map. ILAR 39 (2): 177.
The canine genetic map is in its infancy, although rapid progress is currently in progress. Below are some statements that seemed interesting and might be worth remembering:
1. Chromosome numbers vary within the family Canidae, with 2n=36 in the red fox (Vulpes vulpes) to 2n=78 in the domestic dog and Gray wolf (Canis lupus).
2. Domestic dog and gray wolf share great similarity at mitochondrial DNA level, with 0.2% variation between the species. In contrast, the gray wolf and coyote (Canis latrans) are less closely related with 4% variation at mitochondrial DNA.
3. Most of the approximately 350 identified inherited disorders of the dog are simple recessive traits. Many are homologous to human inherited diseases, for example progressive retinal atrophy (PRA) is equivalent to human retinitis pigmentosa (RP).
4. Majority of inherited disease of the dog are not characterized at the molecular level.
5. Cytogenetic studies in the dog have been constrained by the complex karyotype that comprises 38 pairs of acrocentric autosomes. A standard karyotype for chromosomes 1-21 has recently been established.
The fox gene map. ILAR 39 (2): 182.
Reasons for mapping the fox genes:
Within the Canidea superfamily, the karyotypes are extremely variable with respect to chromosome number and morphology. 2n numbers range from 34 to 78 amoung different canid species. These features are mostly conserved in Feloidea (the other superfamily of Carnivora).
However, the GTG banding patterns are conserved amoung canids but are strongly different from the Feloidea.
These differences can used in comparative genetic analyses with the cat, mink and even humans to provide insights on evolution and gene localization.
Hamster-Fox Somatic Cell hybrids have provided the most information on the fox map.
Intergenic Fox Hybrids (Alopex lagopus x Vulpes vulpes) have been used to map the G6PD gene to the X chromosome. This is how it's done: The enzyme consists of more than one subunit which is expressed by multiple alleles (isoenzymes). Therefore, the enzyme in heterozygotes would normally consist of a combination of subunits contributed by each parent. If the isoenzyme gene is located on the X chromosome, no hybrid isoenzyme will be formed due to inactivation of one of the X chromosomes in somatic cells.
Another interesting feature about the fox is that their numbers of B chromosomes can vary from animal to animal. The B Chromosomes are small, uni- or bi- armed chromosomes. Variation in B chromosome number amoung cells from the same animal has be found. The B chromosome function and origin is poorly understood.
1. In the fox small chromosomes whose number can vary amoung animals are called ? Chromosomes.
Answer = B
The american mink gene map. ILAR 39 (2): 189.
Reviewer's note: Most of this article was "techno-talk" and went into a great deal of genetic minutiae summary focuses on concepts. The American mink (Mustela vison) belongs to the order Carnivora and the family Mustelidae. Carnivores are of great interest in comparative gene mapping, but supplementary data is needed to determine true homology amongst mammals and to reach reliable conclusions about mammalian chromosome evolution. At the time of this article, approximately 30 mink coat color mutations had been identified. Most coat color mutations are controlled by independent autosomal loci. There has only been 1 description of linkage of 2 coat color mutations, Ebony (Eb) and royal pastel (b). The state of mink gene mapping at the time of this article was based on data obtained from traditional breeding, somatic cell hybridization, chromosome-mediated or interphase nuclei-mediated gene transfer procedures, and in situ hybridization.
Chromosomal localization of mink genes was based mainly on 2 panels of clones: mink-Chinese hamster and mink-mouse hybrid cells. Segregation analysis of mink chromosomes and hybrid cell markers made it possible to assign mink genes to particular chromosomes. In mink, 21 genes have been regionally mapped on chromosomes 1, 2, 8, and X. See Figure 1 (pp. 190-191) for mink gene map. The different rearrangements of mink chromosome 2 have been identified by cytogenetic analysis of 2 mink cell lines (MV and MVTK cells) and a mink-Chinese hamster hybrid clone. The MVTK- cells (mink cells deficient in thymidine kinase) were fused with mouse hepatoma cells, and primary hybrid clones were isolated. MVTK- cells contain an intact chromosome 2 and a chromosome 2 containing a small deletion. The development of the in situ hybridization technique offered an efficient technique for regional gene assignment. The isolation of DNA fragments homologous to human or mouse genes from mink DNA libraries provides probes for gene assignment by this technique. Gene mapping by combining somatic cell hybridization and breeding tests has allowed for the identification of various mink gene polymorphisms.
At the time of this article, 77 genes mark all mink chromosomes except the Y, making it possible to compare the mink map with other mammalian genetic maps. The comparison shows that there are >10 large associations of syntenic genes common to mink, human, mouse, and other mammalian species. Synteny is the presence together on the same chromosome of two or more gene loci whether or not in such proximity that they may be subject to linkage. Some syntenic groups of mink genes are characteristic of 3 or more mammalian orders. New data on comparison of mink and human chromosomes using the zoological fluorescence in situ hybridization (ZOO-FISH) technique is available. Specific DNA probes for 22 human autosomes cross-hybridized with 32 large regions of mink chromosomes. In some cases, chromosomal DNA probes showed positive staining on only 1 mink chromosome region whereas in other cases, mink chromosomes were painted by probes derived from 2 or 3 different human chromosomes. There is significant evidence concerning the existence of large conserved regions in mink common to human and mouse as well as other mammalian species. The nature of the conservation of large syntenic gene associations during mammalian evolution is unknown, but the existence of such conserved regions may be interpreted as a result of natural selection.
Syntenic disruption rates during evolution differ significantly among mammalian lineages. The syntenic disruption rate in the mink lineage is moderate and similar to that in baboons, chimpanzees, and cattle. However, it is unclear whether the syntenic disruption rate is constant in the lineage or is possibly higher during the origin of higher taxonomic categories (family, order) than in late speciation within taxa. A syntenic group specific to mink has been found. A comparative analysis of the Geimsa banding with trypsin patterns of chromosomes of >20 species representing 6 genera of the Mustelidae family revealed that all of them possess a chromosome similar to mink chromosome 14. Based on the existence of conserved regions of syntenic genes in phylogenetically distant mammalian species, comparative mapping data may be used to search for important genes in fur bearing mammals.
FYI mink is an ACLAM tertiary species. Here are some mink model questions for review.
1. Transmissible mink encephalopathy is caused by what etiological agent and is an animal model for what human disease? What other animal model is used for this human disease?
2. What disease in mink is used as a model for immunologically mediated glomerulonephritis and arteritis dysgammopathies?
3. What disease in mink is used as a model of tyrosinemia in humans? What are some key differences between this mink model and the human disease?
4. What form of muscular dystrophy do mink display?
1. Etiological agent prion. Human disease - Creutzfeldt-Jacob disease. Other animal model scrapie in sheep. AFIP Fascicle #20 (S).
2. Aleutian disease of mink. AFIP Fascicle #33.
3. Pseudodistemper of mink is an autosomal recessive disorder characterized by primary tyrosinemia type II due to a deficiency of hepatic tyrosine aminotransferase. The clinical syndrome in mink is similar to humans except a) that it is lethal in mink; b) it cannot be controlled by a low tyrosine, low phenylalanine diet; and c) mink do not develop the mental retardation seen in children. AFIP Fascicle # 231.
4. Amyotonic form similar to human amyotonic form. AFIP Fascicle #119.
The common shrew gene map. ILAR 39 (2): 195.
The order Insectivora includes shrews, hedgehogs, moles and tenrecs. It is the 3rd most speciose eutherian order (after Rodentia and Chiroptera) and includes the shrew family Soricidae. The common shrew, Sorex araneus, was chosen as the "type" species of the Insectivora for construction of a gene map. Shrews have a karyotype which includes XX/XY1Y2 system of sex chromosomes and an extremely variable complement of autosomes. Some of the chromosomes of the shrew began as acrocentric chromosomes but fused at the centromeres to form single metacentric chromosomes. The common shrew is subdivided into 50 different karyotypic races. In this study, the Novosibirsk karyotype race was used. Mapping data were obtained from analysis of segregating biochemical markers and shrew chromosomes in the panel of shrew-rodent hybrid clones. The genetic map of the common shrew currently contains 38 genes. Gene content of the X chromosome is well known to have been strongly conserved during the radiation of eutherian mammals. There are 13 syntenic gene associations that have been found to be linked in several species of eutherian mammals of different orders with 6 of the syntenies common to species like primates, rodents and carnivores. Shrew gene mapping in the future should include chromosome painting by fluorescence in situ hybridization with chromosome-specific libraries. The gene map of the common shrew and other information of S. araneus is available at http://meiosis.bionet.nsc.ru/isacc/isacc.htm.
Gene maps of marsupials. ILAR 39 (2): 203.
INTRODUCTION Increased interest in the structural characteristics of mammalian genomes, together with rapid advances in mapping technology, have led to the expansion of gene mapping activity in recent years in the mammalian infraclass Metatheria, better known as marsupial mammals. As late as 1988, only 22 marsupial genes had been reported as mapped by any method in any species (a gene is considered mapped if it adheres to any of the following criteria: (1) assigned to a specific chromosome; (2) a member of a linkage group; (3) autosomal in marsupials but known to be X or Y linked in eutherians). Currently, at least 142 loci have been assigned to physical locations or linkage groups in marsupials, and more than 15 species have at least 3 gene assignments. Most important, 2 distantly related species, Macropus eugenii (tammar wallaby) with 70 loci mapped and Monodelphis domestica (gray, short-tailed opossum) with 69 loci mapped, have emerged as focal species for intensive gene mapping studies. The successes of the Human Genome Project and ancillary mapping efforts in mouse and various livestock species have contributed to the growing recognition of the unique value of marsupial genome data for comparative genome analyses. The advent and application of sophisticated physical mapping tools based on DNA hybridization methods, and the emergence and availability of tractable animal resources (highly productive, pedigreed marsupial colonies) for family studies and linkage analyses, have finally enabled meaningful examinations of the organization and evolution of chromosome structures and gene arrangements in species that had previously been inaccessible to traditional genetic analysis. Marsupials in the Mammalian Scheme The class Mammalia has 3 distinct branches: the subclass Prototheria (monotremes) and the infraclasses Metatheria (marsupials) and Eutheria (so-called placental mammals), which together comprise the subclass Theria. Molecular data documenting the earliest radiation of the 3 branches are inconclusive, but traditional considerations of anatomical, reproductive, and developmental features strongly imply that the prototherian lineage diverged from the common metatherian/eutherian line some 160 to 200 million yr ago (MYA). Results from molecular analyses of globin gene sequences suggest that the ancestors of modem marsupial and eutherian mammals arose later, by means of a split in the metatherian/eutherian lineage approximately 140 to 165 MYA. Extensive analysis of DNA hybridization data led Kirsch and others (1997) to place the metatherian/eutherian divergence considerably later, at 101 to 107 MYA. A recent cladistic analysis based on mtDNA sequence data suggests that the eutherian lineage arose first at approximately 130 MYA, leaving a common metatherian/prototherian line that split again some 15 million yr later (approximately 115 MYA). Egg laying sharply differentiates modern prototherians (the platypus and 2 species of echidnas) from all other living mammals. Although marsupials and eutherians share the live-bearing habit, there are important differences in early developmental events that clearly define these latter groups as well. Most obvious is the difference in the relative proportions of in utero versus ex utero development. By contrast to eutherians, marsupial fetuses are born at an extremely early stage of development after a brief gestation. For species in which placental development occurs, it is minimal and of short duration. Marsupials complete the majority of their "fetal" development subsequent to birth, attached to a teat, and often, but not always, within a protective pouch. Thus, whereas eutherian development is gestationally intensive and occurs internally, primarily via the placental attachment, marsupial development is lactationally intensive and occurs external to the mother. Extant marsupials represent less than 6% of the more than 4,600 mammalian species currently recognized. Marsupials are found on both American continents (approximately 83 species) and in Australasia (Australia, Tasmania, New Guinea, and associated small islands; approximately 170 species). They inhabit cool temperate, hot xeric, and tropical habitats and range from tiny, mouse-like predators of the family Dasyuridae to large grazing and browsing herbivores such as kangaroos and other members of the Macropodidae. Their lifestyles parallel nearly all trophic levels and modes found among eutherian groups. Marsupial Genomes Marsupial genomes are similar in size to those of eutherians but are considerably less diverse in chromosome number and structural arrangements. Chromosome numbers range from 2n--10 to 2n=32, but more than 90% of the species examined exhibit 2n=14 to 22. The most common number, 2n=14, is seen in both American and Australasian species from 10 families. In almost every case this 2n=14 "basic complement" (Role and Hayman 1985) comprises 3 large metacentric chromosome pairs, 2 pairs of intermediate-sized metacentrics, 1 small submetacentric chromosome pair, and the sex chromosomes, X and Y. REASONS FOR MAPPING THESE SPECIES Of little agricultural or commercial importance, marsupials have insufficient economic significance to justify gene mapping research for genetic improvement purposes. Similarly, the perennial absence of a marsupial equivalent of the laboratory mouse has long precluded the intensive use of marsupials in many kinds of basic research, thus obviating the need to map any such species for enhancement of its research potential. Consequently, the primary impetus for genetic mapping studies in marsupials has traditionally related to comparative analyses of gene locations in marsupial genomes relative to those on the detailed maps of eutherian species (particularly human and mouse). The immature state of the newborn marsupial, together with the extended lactational developmental period, facilitates many kinds of fetal manipulations that are impractical or impossible with eutherian embryos. For example, marsupials are used extensively as models for studying normal and regenerative development of the central nervous system and peripheral nervous structures and normal and chemically perturbed skin development, development of endocrine secretion and receptor systems, chemical and ultraviolet radiation-induced skin and eye carcinogenesis, normal and experimentally disturbed patterns of secondary sexual differentiation (for example, Lucas and others, and much more. Successful colonies of 2 species now comprise the fastest growing group of marsupials used for basic biomedical and genetic research purposes. In Australia, the tammar wallaby (M. eugenii, a member of the kangaroo family: Macropodidae) has emerged as the preeminent model marsupial for studies of reproduction, fetal development, lactation, and a variety of genetically related topics including X-chromosome inactivation and the molecular basis and evolution of sex determination. Despite its success as a research model, physical and reproductive limitations related to its size (4.5 to 8.5 kg) and relatively low fecundity (litter size of 1; but can be manipulated to produce multiple offspring annually) will preclude the broad distribution and breeding of this species and thus impede its full development as a laboratory animal. The colony is maintained at Maquarie University in Sydney. The gray, short-tailed opossum (M. domestica) is a South American marsupial (family: Didelphidae) that has been fully adapted to laboratory conditions. It is small (100 to 150 g), highly prolific (mean litter size of 8; up to 3 litters annually), and housed and bred in small cages under conditions that compare favorably with those used for laboratory rodents. Initiated from a small founding stock in 1979, the research colony at the Southwest Foundation for Biomedical Research (SFBR; San Antonio, Texas) has been repeatedly infused with new genetic material from natural populations and exhibits very high genetic. Tens of thousands of animals, including members of outbred and partially inbred strains, have been produced from the SFBR colony, and the distribution of experimental animals and breeding stock to other research laboratories has resulted in the establishment of several colonies worldwide, making M. domestica the predominant laboratory-bred research marsupial in the world today. It serves a broad spectrum of basic and biomedical research topics including normal and abnormal physiology, cellular and organismal development, gene regulation, DNA repair, photobiology, evolutionary genetics, and more. The expanding use of M. eugenii and M. domestica in basic research applications has generated a need to explore the fundamental genetic characteristics of these species to enhance their research utility. The establishment of basic gene maps is among the most important objectives. CURRENT MAP STATUS Presently there are multiple gene assignments for 17 marsupial species representing 3 Australasian families (Macropodidae, Dasyuridae, and Phalangeridae) and 1 American (Didelphidae) family. Fifteen species have more than 4 gene assignments each, and 8 have more than 10 genes mapped. This progress in marsupial gene mapping is summarized in Tables 1 and 2. In Table 1, locations are displayed of genes in the 8 best-mapped species from each of the 4 families relative to their homologues on the human map. In Table 2, additional mapping information is listed for species in which a smaller amount of work has been done. Inspection of Table I reveals that the gene maps of M. eugenii and M. domestica exceed those of any other marsupial species in terms of the number of loci mapped. Mapping progress in M. eugenii has proceeded primarily by physical approaches based on in situ hybridization using radiolabeled heterologous and homologous probes and more recently through more sensitive, fluorescence in situ hybridization (FISH) methods. The data are primarily in the form of chromosome or chromosome arm assignments that indicate synteny affiliations, but there is little information on linkage relationships. Because much of this information was accumulated by less sensitive in situ hybridization methods using radiolabeled heterologous probes, gene ordering is still problematic for many loci. Newer assignments based on FISH have yielded increasingly precise localizations, enabling more confident gene ordering. Due to the emphasis on expressed genes, few anonymous sequences have been mapped in this or any other Australian species. In contrast, the majority of mapping information available for M. domestica is in the form of linkage data, and many of these data pertain to anonymous loci detected by random amplified polymorphic DNA (RAPD) and microsatellite approaches. Of the 58 loci presently assigned to autosomal linkage groups, 24 are expressed genes and 34 are anonymous loci. An additional 3 coding genes are known to be X linked and 1 is Y linked. Nesterova and others (1997) recently published the results of the first mapping studies utilizing M. domestica x rodent cell hybrids. Nine genes were assigned to 4 autosomes and the X chromosome by co-occurrence of M. domestica chromosomes and M. domestica-specific isozyme expression in the M. domestica x rodent somatic cell hybrid panel. Unfortunately, there is little overlap between the current linkage data and the physical mapping data, but the provisional assignment of TK to chromosome 5 by Nesterova and others (1997) and the tentative placement of this locus in linkage group (LG1) 3 by linkage analysis (Sokolova and others 1997) define the first probable chromosomal localization of a large syntenic block to the M. domestica genome. APPROACHES USED TO DEVELOP THE MAPS Marsupial DNA fragments are being cloned with increasing frequency. Currently there are 704 marsupial DNA sequences listed in the GenBank database, including 90 for M. eugenii and 76 for M. domestica. Growth in marsupial DNA resources has permitted the utilization of homologous, or at least closely related, DNA probes for gene mapping and thereby encouraged a shift to FISH methods. The FISH approach has improved the specificity and precision of physical mapping in marsupials to the point where gene ordering is now practical and reliable (for example, Toder and others 1996), and has replaced ISHR for gene mapping studies of M. eugenii and other Australian species. It has not yet been applied to M. domestica mapping. Most recently, the availability of advanced in situ hybridization techniques has enabled the application of chromosome painting methods for the analysis of higher level structural rearrangements of marsupial chromosomes (reviewed by Toder and others 1998). This approach has led to novel insights regarding the evolution of marsupial sex chromosomes that were not obvious at the level of individual gene localizations (for example, Toder and others 1997a,b). Biomedical Applications In addition to physical mapping, continued linkage mapping will soon enable the localization of individual genes that are found to contribute to variation in phenotypes related to physiology, development, and disease susceptibility in the M. domestica model. This concept will be especially valuable when linkage data are finally combined with physical mapping approaches to anchor linkage maps on particular chromosomal regions. Attainment of this goal would enable the direct comparison of regions of the M. domestica genome to homologous regions in the genomes of eutherian species-especially humans wherein they may be of use in the study of normal and anomalous developmental processes and susceptibilities to common diseases. USES OF THE MAPS AND ACCESSIBILITY In the interim, our advice for researchers requiring up-to-date information on the state of the gene maps of particular species is to contact the main laboratories involved. For information on the tammar wallaby (M. eugenii), other macropod species, and dasyurid species: Prof. Jennifer A. Marshall Graves, Department of Genetics and Human Variation, La Trobe University, Bundoora, VIC 3083, Australia; Telephone: +613-9479-2589, Fax: +613-9479-2480; E-mail: firstname.lastname@example.org Dr. Des W. Cooper, School of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia; Telephone: +613-9850-8214, Fax: +613-9850-9686; E-mail: email@example.com For information on the status of the M. domestica map: Dr. Paul B. Samollow, Southwest Foundation for Biomedical Research, P.O. Box 760549, San Antonio, Texas 78245-0549, USA; Telephone: 210-674-1410; FAX: 210-670-3317; E-mail: firstname.lastname@example.org CONCLUSION The development of M. domestica as a model for biomedical research may furnish the means to utilize more effectively the features of marsupial ontogeny that have long been coveted by developmental biologists and expand the use of marsupials as models for a broader range of basic research applications. Today, with the sharpening focus of genetic studies on 2 key species, prospects for the development of detailed marsupial gene maps are excellent.
1. What are the 3 distinct branches of the class Mammalia?
2. True or False: Marsupials complete the majority of their "fetal" development subsequent to birth, attached to a teat, and often, but not always, within a protective pouch. Thus, whereas eutherian development is gestationally intensive and occurs internally, primarily via the placental attachment, marsupial development is lactationally intensive and occurs external to the mother.
3. Define metacentric chromosome.HES
4. What does the abbreviation FISH, ISHR, and RAPD stand for?
5. Name the family, genus and species of the gray, short-tailed opossum. (M. domestica) is a South American marsupial (family: Didelphidae)
6. Give the common name for Macropus eugenii.
1.The class Mammalia has 3 distinct branches: the subclass Prototheria (monotremes) and the infraclasses Metatheria (marsupials) and Eutheria (so-called placental mammals)
2. TRUE: Marsupials complete the majority of their "fetal" development subsequent to birth, attached to a teat, and often, but not always, within a protective pouch. Thus, whereas eutherian development is gestationally intensive and occurs internally, primarily via the placental attachment, marsupial development is lactationally intensive and occurs external to the mother.
3. A metacentric chromosome has a centrally placed centromere
4. FISH, fluorescence in situ hybridization; ISHR, in situ hybridization methods using radiolabeled probes; RAPD, random amplified polymorphic DNA
5. The gray, short-tailed opossum (Monodelphis domestica) is a South American marsupial (family: Didelphidae) that has been fully adapted to laboratory conditions. It is small (100 to 150 g), highly prolific (mean litter size of 8; up to 3 litters annually), and housed and bred in small cages under conditions that compare favorably with those used for laboratory rodents.
6. In Australia, the tammar wallaby (Macropus eugenii, is a member of the kangaroo family: Macropodidae)
Gene maps of monotremes (mammalian subclass prototheria). ILAR 39 (2): 225.
Monotremes include the platypus Ornithorhyncus anatinus from Australia and two types of spiny anteaters, the short beaked echidna Tachyglossus aculeatus from Australia and the Nuguini echidna Zaglossus bruijnii from Guinea. These are the egg laying mammals; young are suckled from milk excreted onto the mother's fur. These animals are important in the history of mammals as an ancestral species which diverged from the placental and marsupial mammals approximately 170 million years ago, based on fossil records.
Monotremes are unique genetically in having a complex of several unpaired chromosomes and in formation of chains of chromosomes during mitosis.
The sex chromosomes are also unique: X chromosomes are G-band homologous over their length, there is no female-specific sex chromatin body at interphase, and there is asynchronous DNA replication of the 2 X chromosomes which appears to be confined to the short are. The short arm of the X chromosome pairs with the Y long arm and the Y short arm pairs with an unpaired autosome.
Mapping of the genome of monotremes was undertaken to use monotremes as "a mammalian outgroup, to deduce the evolution of genome of genome organization, function, and control in mammals." The X chromosome in these animals lacks several genes found on placental (eutherian) mammals, these are also missing in marsupials, thus suggesting that genes were added during evolution.
The genes that have been mapped are presented in a chart with Human, Platypus, and Echidna gene locations and article references. Many more platypus genes than Echidna genes have been mapped to date. A single isozyme of PGK gene is seen in echidnas and is autosomal where this gene is X linked in therian mammals.
Of note: instability of rodent-monotreme cell hybrids has limited the effort to map these genes. In situ hybridization has been most successful.
Future studies may help in elucidating the evolutionary origin of the human genome; however, at this time the monotreme information is most applicable to understanding sex chromosome evolution.
1. Which of the features below is not found in monotremes?
b. Mammary gland teats
c. Eggs layed externally
d. mother suckles young
2. Why is the genome of monotremes of importance?
a. For genetic manipulation of these animals
b. For studying the evolution of other mammals
c. It is not important
3. Which chromosomes have been most studied to date in monotremes?
b. X chromosomes
c. Y chromosomes
d. both b and c
e. a, b, and c
1. b, 2. b, 3. d
The chicken gene map. ILAR 39 (2): 229.
Most of the information generated to date on bird genomes has been derived from chickens (Gallus gallus). Primary interests in mapping this species include: study of genetic marker traits of economic value, use of chickens as animal models of quantitative trait loci - QTLs (eg. weight, fatness and disease resistance), genetic disease and developmental defects, and study of the evolution of the vertebrate genome. Study of many economically important QTL traits requires crosses between lines with vastly differing phenotypes and large numbers of progeny (>500 F2 intercross progeny). QTLs (and their encoded genes) are likely to be conserved across species. Research involving these genes would thus be difficult, if not impossible in many larger mammals and humans. Chickens are a convenient species to study because they have a short intergeneration interval, they produce a large number of progeny, rearing costs are low, DNA can be extracted from nucleated erythrocytes, and they have a relatively small genome (1.2 billion base pairs). Two reference mapping populations have been used to generate the genomic map to date - one in the UK and one in the US. These populations have been typed using both expressed gene markers and anonymous markers (eg, random gene clones, minisatellites, endogenous retroviruses, etc) such that almost 1200 marker genes have been mapped. Despite evolutionary divergence over 350 million years previously, there is moderate homology between mammalian and chicken genomes. Comparative mapping studies have linked a single autosomal gene mutation for dwarfism in chickens (ADW, chromosome 1) with a pygmy mutant phenotype in mice resulting from a high mobility group protein mutation on murine chromosome 10. Studies in chickens have been hampered in that microchromosomes are too small to be recognized individually with conventional banding techniques and they have a low number of microsatellite markers compared with other species, for high resolution mapping. Use of other marker types such as amplified fragment length polymorphisms and nucleotide polymorphisms, in addition to FISH techniques and radiation hybrids may permit work to proceed more rapidly in the future. Mapping summaries may be found at: http://www.ri.bbsrc.ac.uk/
1. What is the diploid chromosomal complement for chickens?
2. What are the avian designations for the sex chromosomes?
2. Sex chromosomes are designated as Z and W.
The linkage map of Xiphophorus fishes. ILAR 39 (2): 237.
Despite significant diversity between species, over 50% of all bony fish examined to date have a diploid chromosomal complement of 48. There are less gene rearrangements in fish compared with mammals, suggesting that fish may retain more genetic homology to a common ancestor of all vertebrates, than other classes of vertebrates. Fish are therefore often used for comparative gene mapping studies. Comparative gene mapping assists in exploring i) how chromosomes and particular gene arrangements function, ii) the constraints imposed on chromosomal evolution by natural selection and iii) the relationship between speciation and rates and types of chromosomal rearrangements. In addition, gene mapping in Xiphophorus fishes has been performed to explore the function of oncogene and tumour suppressor gene regulation of a spontaneous malignant melanoma which occurs in these fish (ie. cancer initiation and progression). Xiphophorus fish are livebearers and thus egg/embryo manipulation is not available/difficult to perform for genetic studies The current map is a composite obtained from multiple crossings of 15 species within the genus. To date, 334 genetic markers have been identified, including 104 genes. Mapping summaries may be obtained from: http://sprd1.mdacc.tmc.edu/skazianis/mainpage.html
1. Define teleost.
2. Which 3 species of fish have been used most commonly for chromosomal mapping studies?
1. a bony fish
2. zebrafish (Danio rerio), platyfishes & swordtails (Xiphophorus), medaka (Oryzias latipes)
The pufferfish gene map. ILAR 39 (2): 249.
The earliest true vertebrates are represented by fish. The largest class of fish is the teleost, or bony fish. The genomic organization of fish differs in several significant ways from mammals. Fish chromosomes are much smaller, they do not show Giemsa banding, and have a higher frequency of the dinucleotide CpG.
Classical linkage studies have not been performed in the pufferfish but there has been an emphasis on studying the physical genome with particular emphasis on comparative gene structure and organization. The haploid genome size is approximately 400 Mb. Although much smaller than mammalian genomes, the gene number and gene structure are similar. Due to the large evolutionary gap (400 million years), between teleosts and mammals, the only elements that appear to be conserved are the essential o nes. This has allowed identification of regulatory elements by comparative sequence analysis.
Assessment of the Fugu genome has been carried out at the sequence level by sequence scanning techniques. With the high degree of similarity between Fugu and mammalian genes at the nondegenerate amino acid level and the high gene density, sequence scanning of genomic clones is a relatively easy and efficient way to identify genes in Fugu. It has been possible to identify many genes and to acquire close-range physical linkage data, even though the gene order may not be accurate.
Sequence scanning is a relatively new technique. It has limited use with very large genomes such as human and mouse. It relies on database searches to define hits on genes, and has been costly until recent availability of more robust sequencing enzymes. It provides an overall picture of gene content and organization. Limitations include the lack of absolute gene order and the ability to correlate the data directly to traditional maps as well as the fact that genes are present only in fragments. All data from the Fugu genome project are available on a web site and users may search by DNA, amino acid sequences, or by key words.
An increasing number of genes are being sequences in a variety of fish species which will produce meaningful fish maps. Fugu genomics will be useful in contributing to gene finding, sequencing, and functional characterization of fish genetics.
1. Give the family, genus and species of the pufferfish.
2. Give the genus and specis of the zebrafish.
3. What are pufferfish most commonly used for?
1. Tetraodontiformes, Fugu rubripes
2. Danio rerio
3. They are farmed commercially in Japan and eaten as a delicacy.