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ILAR Journal V39(2/3) 1998
Comparative Gene Mapping
Jennifer A. Marshall Graves
| Jennifer A. Marshall Graves, Ph.D., is Professor and Head of the School of Genetics and Human Variation, La Trobe University, Bundoora, Australia. |
INTRODUCTION
Monotremes comprise a single order representing the mammalian subclass Prototheria. Distinguished by a mode of reproduction unique among mammals--laying eggs they incubate in burrows--monotremes suckle their young with milk licked from the fur. Fossil evidence and anatomy, as well as their mode of reproduction and molecular evidence, suggest that they diverged from therian mammals ("placental" eutherian and marsupial mammals) approximately 170 million yr ago (Hope and others 1990; Kemp 1982), although recent analysis of the entire mitochondrial genome has resulted in some doubt regarding the order of divergence (Janke and others 1996).
Only 3 species of monotremes exist in 2 families variously considered to have diverged 30 to 70 million yr ago (Westerman and Edwards 1990). The platypus Ornithorhynchus anatinus is widespread in eastern Australia but vulnerable because of its aquatic habitat. Two species of echidna (spiny anteater) are the short-beaked echidna Tachyglossus aculeatus in Australia and the closely related Nuguini echidna Zaglossus bruijnii in New Guinea. The platypus has 2n=52, whereas the 2 echidna species (with G-band identical karyotypes) have 2n=63 (male) and 64 (female) (Murtagh 1977; Watson and others 1992c; Wrigley and Graves 1988a).
Monotremes are unique among vertebrates in possessing a complex of several unpaired chromosomes that form a chain with the sex chromosomes at male meiosis. The short arm of the X pairs with the long arm of the Y, the short arm of the Y then pairs with another chromosome, it in turn pairs with another chromosome, and so on. Thus, several elements (9 in echidna and 8 in the platypus) are observed at meiosis arranged end-to-end as the result of terminalization of chiasmata. The autosomes in the chain are observed at mitosis as small elements without homologous pairs. The multivalent chain was presumably formed by serial translocations between sex chromosomes and autosomes. The interpretation of this chain has been the subject of several studies (Bick 1992; Watson and others 1992a) and is still not fully resolved because of the small size and poor banding of the autosomes, in addition to difficulties in obtaining material.
Monotreme sex chromosomes are of particular interest. In all 3 species, the X chromosomes are G-band homologous over their length, and homology has been confirmed by gene mapping (Watson and others 1992a,b; Wrigley and Graves 1988a). It is still not clear whether the X is subject to inactivation. No female-specific sex chromatin body is visible at interphase (McKay and others 1987). Asynchronous DNA replication of the 2 X chromosomes in females (at least in lymphocytes) has been reported; however, asynchrony appears to be confined to the short arm that is paired with the Y and should not require dosage compensation (Murtagh 1977; Wrigley and Graves 1988b). Since the entire long arm of the Y chromosome pairs with the entire short arm of the X (Murtagh 1977), and the Y then pairs over an unknown portion of its short arm with an unpaired autosome, we cannot be certain that the Y contains male-specific DNA, although it is limited to males.
Monotremes are unusual in having sperm with fibrillar nuclei. This has enabled demonstration of the ordered tandem arrangement of chromosomes (Watson and others 1996).
REASONS FOR MAPPING THIS SPECIES
Monotremes are particularly interesting because they are the mammals most distantly related to placental mammals (such as humans and mice) and marsupials. Monotremes therefore serve as a mammalian outgroup. We can usc comparisons of gene arrangement, nucleotide sequence, and gene function to deduce the evolution of genome organization, function, and control in mammals. Comparisons between these distantly related groups can be used to reconstruct the genome of an ancient ancestral mammal that lived more than 170 million yr ago, early in the age of mammals. For instance, the demonstration that the marsupial X chromosome lacks many genes present on the eutherian X could be interpreted either as addition of autosomal material in the eutherian lineage or loss in the marsupial lineage. The finding that the genes are also missing from the X in monotremes identifies this as the ancestral condition and suggests that an autosomal region has been added to sex chromosomes in an ancestral eutherian, an important conclusion with far-reaching implications for mammalian genome evolution (Graves 1995).
CURRENT MAP STATUS
A total of 35 genes have been assigned to platypus chromosomes and 5 to the short-beaked echidna. Locations of these genes are given in Table 1.
APPROACHES USED TO DEVELOP THE MAP
Family studies in these species are all but impossible since the animals do not breed in captivity. However, considerable efforts have been made to reconstruct families from wild-caught samples by DNA fingerprinting and microsatellite and mitochondrial DNA analysis (S. Akiyama, La Trobe University, Bundoora, Australia, personal communication, 1998; Gemmell and others 1994, 1995). Limited population data are also available. Of particular interest is the observation that there is only a single isozyme of the PGK gene in echidnas, and males may be heterozygous at this single locus (which must therefore be autosomal or pseudoautosomal). This contrasts with therian mammals in which the gene is X linked and there is an autosomal expressed homologue that is testis specific (VandeBerg and Cooper 1978).
Despite considerable effort, somatic cell genetic analysis has been limited by the instability of rodent-monotreme cell hybrids (Watson and Graves 1987). Synteny was established by segregation of PGK and HPRT from the few cell hybrids that retain platypus chromosomes and genes, but no chromosome assignment could be made to the small and possibly fragmented platypus chromosomes retained. Recent in situ hybridization shows that PGK and HPRT, as well as some other human X-linked genes, are autosomal in the platypus (J. M. Watson, La Trobe University, personal communication, 1998).
The most successful means of mapping has been in situ hybridization. Using heterologous probes (usually human cDNA) to very conserved genes, physical locations have been obtained for many genes using radioactive in situ hybridization to platypus and echidna chromosomes of cultured cells (Wrigley and Graves 1984). Several genes have now been cloned from the platypus, and fluorescence in situ hybridization using long genomic clones (Wilcox and others 1996) has been used to localize platypus genes (A. Pask, La Trobe University, personal communication, 1998).
SCIENTIFIC CONTRIBUTIONS OF THE MAP
The value of monotreme gene mapping has been demonstrated by studies of mammalian sex chromosomes (Graves 1995; reviewed by Graves and Watson 1991). Comparisons of the genes on the X and Y chromosomes in monotremes with the arrangement in marsupials and eutherians has provided an understanding of mammalian sex chromosome evolution. Genes on the long arm and pericentric region of the human X map to the X also in monotremes (and marsupials), but genes on the short arm of the human X distal to Xp11.23 are autosomal in monotremes (and marsupials). The most simple explanation for these results is that the region of the human X distal to Xp11.23 was not part of the original X but has been added to the X recently in the eutherian lineage. The finding (Graves and Watson 1991) that human Xp genes lie in at least 3 conserved clusters on marsupial and monotreme autosomes demonstrated that the mammalian X chromosome has received multiple additions.
The evolution of the Y chromosome is even more interesting than that of the X. It was suggested decades ago (Ohno 1967) that the mammalian X and Y chromosomes evolved from a pair of autosomes in an ancestral mammal by a process of degradation of the Y. This idea is supported by the presence of the pseudoautosomal region and of paralogous genes on the human X and Y. The location of UBE1 in a range of mammals provides a good demonstration of this postulated degradation (Mitchell and others 1998), with monotremes representing the original condition in which the gene was pseudoautosomal. In contrast, in the differentiated X and Y copies that exist in all eutherian mammals except primates, only inactive fragments of the gene are found on the Y. This progression shows that the gene was originally paired but has been specialized, degraded, or lost on the Y in different eutherian species.
The absence of several of the human XY-shared genes from the marsupial and monotreme X implies that a region was added, not only to the X but also to the Y in eutherians. Thus, the Y chromosome was subject to additions as well as attrition during evolution. An addition-attrition hypothesis has been put forward to explain how autosomal regions can be added to an ancient pseudoautosomal region that subsequently degrades on the Y and becomes incorporated into the X inactivation system on the X (Graves 1995).
Comparative gene mapping between monotremes, marsupials, and eutherians has also provided some information about the evolution of autosomes. For instance, it was found that 5 human chromosome 21 genes lay in the same 2 clusters in monotremes as well as in marsupials, implying that this was their ancestral arrangement (Maccarone and others 1992).
ANTICIPATED FUTURE CONTRIBUTIONS OF THE MAP
Similar studies could help elucidate the evolutionary origin of the rest of the human genome. Details of the evolution of the X and Y chromosomes will be of particular interest, as will identification of the unpaired elements and investigation of their behavior at meiosis and fertilization.
In addition, the cloning and mapping of monotreme genes is likely to assume greater importance in studies of gene function. For instance, the search for a monotreme homologue of the SRY gene, which is testis determining in humans and mouse (and probably marsupials), has revealed homologues only on autosomes (A. Pask, personal communication, 1998), suggesting that monotreme sex determination does not depend on this gene. It will also be important to establish whether X inactivation occurs in monotremes by examining the expression of genes mapped to the unpaired portion of the X that might require dosage compensation.
USES OF THE MAP AND ACCESSIBILITY
The special value of the monotreme gene map is as a mammalian outgroup. It will be possible to use the map to determine the ancestral arrangement of any given part of the genome in humans or other mammals. This kind of information will make it possible to use conserved synteny for predicting gene location in a new species or for new genes. At the time of this writing, these data are available on the Web (http://www.latrobe.edu.au/www/genetics/roobase.html) in the form of a table entitled "Roobase."
CONCLUSION
Monotremes consist of a small group of fascinating mammals that are especially valuable because of their distant relationship with the usual model mammals, mouse and human. Because of this relationship, monotremes are likely to provide more variation of even extremely conserved gene arrangements and genetic control mechanisms. Comparisons of gene sequence, arrangement, and function between monotremes and therian mammals can afford us unique information about early events in the evolution of the genome and genetic control systems in mammals.
ACKNOWLEDGMENTS
The paramount contribution of Dr. Jacki Watson (Wrigley) throughout her brilliant and all too brief career dedicated to monotreme genetics is acknowledged.
REFERENCES
Bick YAE. 1992. The meiotic chain of chromosomes of Monotremata. In: Augee M, editor. Platypus and Echidnas. Sydney NSW Australia: Royal Soc. NSW. p 64 68.
Gemmell NJ, Grant TR, Western PS, Watson JM, Murray ND, Graves JAM. 1995. Determining platypus relationships. Aust J Zool 43:283-291.
Gemmell NJ, Janke A, Western PS, Watson JM, Paabo S, Graves JAM. 1994. Cloning and characterization of the platypus mitochondrial genome. J Mol Evol 39:200-205.
Graves JAM. 1995. The origin and function of the mammalian Y chromosome and Y-borne genes--An evolving understanding. Bioessays 17:311 320.
Graves JAM, Watson JM 1991. Mammalian sex chromosomes: Evolution of organization and function. Chromosoma 101:63-68.
Hope R, Cooper S, Wainwright B. 1990. Globin macromolecular sequences in marsupials and monotremes. Aust J Zool 37:289-313.
Janke A, Gemmell NJ, Feldmaier-Fuchs G, von Haeseler A, Paabo S. 1996. The mitochondrial genome of a monotreme--The platypus (Ornithorhynchus anatinus). J Mol Evol 42:153-159.
Kemp TS. 1982. Mammal-like Reptiles and the Origin of Mammals. New York: Academic Press.
Maccarone P, Watson JM, Francis D, Kola l, Graves JAM. 1992. The evolution of human chromosome 21: Evidence from in situ hybridization in marsupials and a monotreme. Genomics 13:1119-1124. McKay LM, Wrigley JM, Graves JAM. 1987. Evolution of mammalian X chromosome inactivation: Sex chromatin in monotremes and marsupials. Aust J Biol Sci 40:397-404.
Mitchell M J, Wilcox SA, Lerner J, Woods D, Scheffler J, Hearn J, Bishop C, Graves JAM. 1998. The origin and loss of the ubiquitin activating enzyme (UBEI) genes on the mammalian Y chromosome. Hum Mol Genet 7:429-434.
Murtagh CE. 1977. A unique cytogenetic system in monotremes. Chromosoma 65:37-57.
Ohno, S. 1967. Sex Chromosomes and Sex Linked Genes. Berlin: Springer-Verlag.
Sinclair AH, Wrigley JM, Graves JAM. 1987. Autosomal assignment of OTC ill marsupials and monotremes: Implications for the evolution of sex chromosomes. Genet Res 50:131-136.
Spencer JA, Watson JM, Lubahn DB, Joseph DR, French FS, Wilson EM, Graves JAM. 1991. The androgen receptor gene is located on a highly conserved region of the X chromosomes of marsupial and monotreme, as well as eutherian mammals. J Hered 82:134-139.
VandeBerg JL, Cooper DW. 1978. Possible autosomal inheritance of erythrocyte phosphoglycerate kinase A in echidnas. Biochem Genet 16:1031 -1034.
Watson JM, Collett C, Westerman M, Graves JAM. 1991a. Localization of autosomal gene homologues in the monotreme mammal Ornithorhynchus anatinus (the platypus) (Abstract). Cytogenet Cell Genet 58:2131.
Watson JM, Frost C, Spencer JA, Graves JAM. 1993. Sequences homologous to the human X-and Y-borne zinc finger protein genes (ZFX/Y) are autosomal in monotreme mammals. Genomics 15:317-322.
Watson JM, Graves JAM. 1987. Gene mapping in marsupials and monotremes. V. Synteny between hypoxanthine phosphoribosyltransferase and phosphoglycerate kinase in thc platypus. Aust J Biol Sci 41:231-237.
Watson JM, Meyne J, Graves JAM. 1992a. Studies of the chromosomes of the echidna meiotic translocation chain. In: Augee M, editor. Platypus and Echidnas. Sydney NSW Australia: Royal Soc. NSW. p 53-63.
Watson JM, Meyne J, Graves JAM. 1996. Ordered tandem arrangement of chromosomes in sperm heads of monotreme mammals. Proc Natl Acad Sci U S A 93:10200 10205.
Watson JM, Riggs AD, Graves JAM. 1992b. Gene mapping studies confirm the homology between the platypus X and echidna X I chromosomes and identify a conserved ancestral monotreme X chromosome. Chromosoma 1 (11:596-601.
Watson JM, Spencer JA, Graves JAM, Snead ML, Lau EC. 1992c. Autosomal localization of the amelogenin gene in monotremes and marsupials: Implications for mammalian sex chromosome evolution. Genomics 14:785-789.
Watson JM, Spencer JA, Riggs AD, Graves JAM, 1990. The X chromosome of monotremes shares a highly conserved region with the eutherian and marsupial X chromosomes, despite the absence of X chromosome inactivation. Proc Natl Acad Sci U S A 87:7125-7129.
Watson JM, Spencer JA, Riggs AD, Graves JAM. 1991b. Sex chromosome evolution: Platypus gene mapping suggests that part of the human X chromosome was originally autosomal. Proc Natl Acad Sci U S A 88:11256-11260.
Westerman M, Edwards D. 1990. The divergence between echidna (Monotremata: Tachyglossidae) and platypus (Monotremata: Ornithorhynchidae)--New data from DNA studies. Aust Mamm 14:115-120.
Wilcox SA, Toder R, Foster JW. 1996. Rapid isolation of recombinant lambda phage DNA for use in fluorescent in situ hybridization. Chromosome Res 4:397-398.
Wrigley JM, Graves JAM. 1984. Two monotreme cell lines, derived from female platypuses (Ornithorhynchus anatinus; Monotremata, Mamma-Ira). In Vitro 20:321-328.
Wrigley JM, Graves JAM. 1988a. Karyotypic conservation in the mammaIian order Monotremata (subclass Prototheria). Chromosoma 96:231-247.
Wrigley JM, Graves JAM. 1988b. Sex chromosome homology and incomplete, tissue-specific X-inactivation suggest that monotremes represent an intermediate stage of mammalian sex chromosome evolution. J Hered 79:115-118.
TABLE 1 Locations of genes in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus)a
| Gene symbol | Gene name | Location | Reference | ||
| Human | Platypus | Echidna | |||
| RAF | Murine leukemia viral (v-raf) oncogene homologue | 3p25 | 1p(proxb) | Watson and others 1991a | |
| MYB | Avian myeloblastosis viral (v-myb) oncogene homologue | 6q22-23 | 2p (prox) | Watson and others 1991a | |
| TUBB | Tubulin, beta | 8pter-q21 | 3p (distb) | Watson and others 1991a | |
| HBB | Hemoglobin B | 11p15.5 | 4q (dist) | Watson and others 1991a | |
| IG@ | Immunoglobulin G, M, E | 14q32.33 | 5q (midb) | Watson and others 1991a | |
| SOD1 | Superoxide dismutase | 21q22.1 | 4q (mid) | Maccarone and others 1992 | |
| CBR | Carbonyl reductase | 21q22.1 | 4q (mid) | Maccarone and others 1992 | |
| BCE1 | Breast cancer estrogen-induced 1 | 21q22.2 | 4q (mid) | Maccarone and others 1992 | |
| INFAR | Interferon alpha receptor | 21q2.2 | 2q (mid) | Maccarone and others 1992 | |
| ETS2 | Avian leukemia E26 viral proto-oncogene | 21q22.3 | 2q (mid) | Maccarone and others 1992 | |
| PDGFB | Platelet-derived growth factor beta | 22q12.3-q13.1 | 4p | Watson and others 1991a | |
| AMEL | Amelogenin | Xp22.21-22.1/Yq | 2q (dist) | Watson and othes 1992c | |
| POLA | DNA polymerase alpha | Xp22.1-21.3 | 2p (mid) | Watson and others 1991b | |
| ZFX/Y | Zinc finger on X/Y | Xp/Yp | 1q (prox) | Watson and others 1993 | |
| DMD | Dystrophin | Xp22.1-21.3 | 1q (prox) | Watson and others 1991b | |
| CYBB | Cytochrome b-245, beta chain | Xp21.1 | 1q (prox) | Watson and others 1991b | |
| OTC | Ornithine transcarbamylase | X[21.1 | 2p (mid) | Sinclair and others 1987 | |
| MAOA | Monoamine oxidase A | Xp11.4-.23 | 1q (dist), 2q (prox) | Watson and others 1991b | |
| TIMP | Trypsin inhibitor of metalloprotease | Xp11.3-.23 | 1q (dist) | Watson and others 1991b | |
| SYN | Synapsin | Xp11.2 | 2p (mid) | Watson and others 1991b | |
| UBE1X | Ubiquitin binding enzyme 1 (Y copy in mouse) | Xp11.3-11.23 | Xp (dist)Yq | Mitchell and others 1998 | |
| ARAF1 | Proto-oncogene ARAF1 | Xp11.3-11.23 | 2q (dist) | Watson and others 1991b | |
| AR | Androgen receptor | Xq12 | Xq (prox) | Xq (prox) | Spencer and others 1991 |
| PGK1 | Phosphoglycerate kinase 1 | Xq13.3 | U1 | Watson and Graves 1987 | |
| GLA | Galactosidase alpha | Xq21.3-q22 | Xp (prox) | Watson and others 1990 | |
| PLP | Proteolipid protein | Xq21.3-q22 | Xp (prox) | Watson and others 1990 | |
| HPRT | Hypoxanthine phosphoribosyltransferase | Xq26.1 | U2 | Watson and Graves 1987 | |
| F9 | Coaggulation factor IX | X26.3-q27.1 | Xp (prox) | Xp (prox) | Watson and others 1990 |
| MCF2 | Unknown oncogene | Xq26.3-q27.2 | 2q, Xq (dist) | 2q, Xq (dist) | Watson and others 1990 |
| F8 | Coaggulation factor VIII | Xq28 | Xp (prox) | Watson and others 1990 | |
| G6PD | Glucose-6-phosphate dehydrogenase | Xq28 | Xq (dist) | Xq (dist) | Watson and others 1990 |
| GDX | Unknown gene | Xq28 | Xq (dist) | Xq (dist) | Watson and others 1990 |
| P3 | Unknown gene | Xq28 | Xq (dist) | Watson and others 1990 | |
| RCP | Red cone pigment | Xq28 | Xp (prox) | Watson and others 1990 |
bProx, proximal; dist, distal; mid, medial.
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