ILAR Journal Online, Volume 39(2/3) 1998: Comparative Gene Mapping

Online Issues

<< All Back-issues

<< This Issue's Table of Contents

ILAR Journal V39(2/3) 1998
Comparative Gene Mapping

The Vole Gene Map
T. B. Nesterova, N. A. Mazurok, N. V. Rubtsova, A. A. Isaenko, and S. M. Zakian

T. B. Nesterova, Ph.D., N. A. Mazurok, Ph.D., N. V. Rubtsova, M.S., A. A. lsaenko, M.S., and S. M. Zakian. D.Sci., are with the Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Department, Novosibirsk, Russia. T. B. Nesterova is also with the MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom.

INTRODUCTION

Gray voles belong to the Arvicolidae family and, together with mouse and rat, to the vast Muroidea superfamily. All representatives of the genus Microtus look quite similar; a nonspecialist can see hardly any differences between them, except perhaps their slight variations in size and fur color. Voles are small rodents, with body sizes ranging between 80 and 145 mm, depending on species and sex. They have short tails (approximately one third their body length) with light gray to dark brown fur (Meyer and others 1996). They can be maintained in captivity relatively easily, and the conditions reported previously are suitable (Jackson 1997).

The taxonomic status of different species of genus Microtus has been revised many times, and the exact status of some species of voles is still questionable. The relative rank of voles is complicated by the high morphological similarity of different representatives of gray voles, which led to misidentification of species using zoological criteria. The karyological approach shed more light on vole taxonomy and allowed identification of groups of species instead of a single polytypical species. This article describes the result of our efforts in mapping the genomes of 5 species of gray voles: M. arvalis, M. rossiaemeridionalis, M. kirgisorum, M. transcaspicus, and M. agrestis.

Many revisions have been made to the status of Microtus arvalis (common vole). Initial, unsuccessful attempts to describe its chromosome set date back to the late 1930s, when Renaud first reported the correct number as 2n=46 (Renaud 1938). Additional research on the species has revealed many chromosome forms that had previously been considered subspecies.

In 1965, the chromosome sets of voles were studied from subspecies M. arvalis duplicatus captured in the Leningrad region of Russia, and the diploid number was identified as 54, not 46, chromosomes (Meyer and others 1967). Later, sympatric sibling species were reported (Meyer and others 1972). The 46-chromosome vole has retained the M. arvalis name, although its 54-chromosome mate was initially named M. subarvalis (Meyer and others 1972) and then renamed M. rossiaemeridionalis (Malygin 1983).

Discovery of the sympatric sibling species by cytogenetic analysis demanded total taxonomic revision of this polytypical species. Further karyological and hybridization analysis provided evidence that the Transcaspian (M. transcaspicus) and Kirgizian (M. kirgisorum) voles each comprise a species of its own (Meyer and others 1971; Malygin 1973). However, M. orcadensis, M. asturianus, M. sarnius, M. incertus, and M. igmanensis, once regarded as different species, have been identified as subspecies within M. arvalis. Furthermore, 2 chromosome forms--arvalis and obscurus--have been identified in M. arvalis (Orlov and Malygin 1969). Different localization of the centromeres in a few pairs of small autosomes and the Y chromosome led to different numbers of arms in these karyoforms. Populations with 6 or fewer pairs of acrocentric chromosomes are combined in the arvalis group; those with 9 and more in obscurus. Each of the karyoforms consists of numerous subspecies and inhabits a vast area. Arvalis is distributed throughout Europe; and obscurus, throughout Siberia, Urals, and Caucasus. Geographically, both forms border each other but never overlap (Orlov and Malygin 1969; Meyer and others 1972). Whether they are different species or still undergoing speciation is unresolved at the time of this writing.

Cytogenetic analysis has thus revealed that M. arvalis is not the only polytypic species but is instead a group of at least 4 relative species. M. kirgisorum and M. transcaspicus live allopatrically in Middle Asia, whereas M. arvalis and M. rossiaemeridionalis are sympatric sibling species widely spread in Europe.

Systematically, M. agrestis is the closest to the arvalis group, with a diploid chromosome number of 50 (Cooper and Hsu 1972). The species is polytypic and combines more than 20 subspecies forms (Meyer and others 1996). M. agrestis 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.

REASONS FOR MAPPING THIS SPECIES

Because of the specific pattern of its distribution and systematic and chromosome differentiation, the arvalis group is a valuable experimental model for evolutionary, phylogenetic, and genetic studies. We have used these voles as laboratory animals in studies of the mechanism of X inactivation (Zakian and others 1987, 1991 ). This experimental model is advantageous because of the unusual pattern of X chromosome inactivation occurring in hybrids from different crosses. Although interspecific hybrids of most vole species undergo normal random X inactivation (that is, an equal probability of either X chromosome to be inactivated in a given female cell), hybrids with M. arvalis preferentially inactivate the X chromosome of the other parent. Understanding the molecular basis of this phenomenon is impossible without knowing the genetics of the species under study and especially without having detailed cytogenetic maps of their genomes.

Mapping the 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. At the time of this writing, research mapping the X chromosomes is of particular interest since those maps will make it possible to compare 5 related species with mouse and human. The data obtained could be useful in reconstructing the ancestral mammalian X chromosome.

CURRENT MAP STATUS

The cytogenetic maps of common vole genomes are represented by rRNA loci, corresponding to the nucleolus organizer regions (NORs¹), and by 5 genes assigned to the X chromosome. The M. agrestis cytogenetic map contains data for X-linked genes only.

Localization of NORs

A total of 16 autosomes with NORs have been identified in M. rossiaemeridionalis (Figure 1) and 11 autosomes in M. transcaspicus (Figure 2). NORs are located near the centromere on the long arm of acrocentric chromosomes in both species. When stained routinely, the zones where functionally active NORs are located are visible as secondary constrictions, whose variable size depends on the corresponding region's level of activity. NORs are located in the short arms of 13 acrocentric autosomes and in the short arm of the acrocentric X chromosome in M. kirgisorum (Figure 3).

Ten autosomes with NORs have been identified in M. arvalis obscurus (Figure 4a) and 5 in M. arvalis arvalis (Figure 4b). In M. a. obscurus, NORs are located in the short arms of 6 acrocentrics and in the telomeric regions of the short arms of the 4 largest metacentrics. In M. a. arvalis, NORs are located in the short arms of 4 acrocentrics and in the telomeric region of the short arm of a medium-sized metacentric.

In the idiogram of G-banded chromosomes obtained with trypsin treatment (GTG¹)(Figures 1 through 4), these regions are designated according to the standard nomenclature for human chromosomes (ISCN 1981 ). This nomenclature is convenient, although it is important to remember that the band on the chromosome may vary in size or be missed totally, depending on the level of corresponding NOR transcriptional activity.

Mapping the X Chromosome

Five genes have been assigned to the X chromosomes in 5 Microtus species from the arvalis and agrestis groups: Hprt (hypoxanthine phosphoribosyl transferase), Pgk1 (phosphoglycerate kinase 1), Gla (a-galactosidase), and G6pd (glucose-6-phosphate dehydrogenase), which are constitutively expressed housekeeping genes; and Xist (X inactive specific transcript), which is a master regulator of X chromosome inactivation (Penny and others 1996). The positions of the 5 genes in the 4 arvalis species and in M. agrestis are illustrated on gene maps of their X chromosomes (Figures 1 through 5).

Pgk1, Xist, and Gla localize in a region with a similar Giemsa (G)-banding pattern in all 5 species of voles. Pgk1 and Xist are the most tightly linked genes in the whole group and are separated from Gla by at least 1 G-dark band.

G6pd and Hprt have been assigned to the 2 G-light bands separated by one G-dark band in all vole species. Slight differences in the G-banding pattern in the Xq telomeric region in M. arvalis and M. kirgisorum, compared with the other species, suggest that an inversion has occurred in the ancestral vole X chromosome. This suggestion is consistent with our mapping data (Nesterova and others 1998).

APPROACHES USED TO DEVELOP THE MAP

We used several approaches to localize X-linked genes. The X-linkage of G6pd and Gla in common voles was first established in an analysis of gene expression in reciprocal hybrids among 4 species (Zakian and others 1987, 1991 ). However, this method was not very productive for our purposes since we could find neither inter- nor intraspecific polymorphism for any other genes to check whether they also were X-linked. To prove the X-linkage of Hprt and Pgk1, we used somatic cell hybridization between mouse and vole fibroblast cells. Concordant segregation of Hprt, Pgk1, G6pd, and Gla with vole X chromosome has demonstrated their X-linkage unambiguously (Nesterova and others 1994). Nevertheless, because the methods described above have not revealed the position of the genes on the chromosome, attempts to create a cytogenetic map continue at the time of this writing.

To establish a cytogenetic map, one should be able to distinguish between different chromosomes in the set and to determine a particular region on the chromosome where a gene of interest is located. For the vole, cytogenetic information was relatively scarce, and G-banding of moderately coiled chromosomes available for some species did not afford a clear identification of all chromosome pairs. Extensive research has been undertaken to obtain high-resolution GTG-banded chromosomes for 4 species of common vole (Mazurok and others 1994, 1995, 1996a,b). More than 400 bands in the haploid set of chromosomes have been defined for each species, leading to reliable identification of any region of vole genome and creating a basis for chromosome and subchromosome mapping.

During the 1990s, fluorescence in situ hybridization (FISH¹)has become a widely used method for mapping genes in different species. We have used this method to regionally localize Gla, G6pd, Hprt, Pgk1, and Xist on the X chromosome in 5 members of the genus Microtus (Nesterova and others 1998). Since the best results are usually obtained with homologous probes, we performed the FISH analysis with vole probes isolated from the vole genomic libraries. Each probe was mapped individually, and 2-color FISH analysis was then used to order the genes on the chromosomes.

To identify the NORs, we used a silver staining method as described by Howell and Black (1980). This method detects nucleolus organizers that were active at the interphase that preceded current mitosis. To identify nucleolus organizer chromosomes, silver staining was followed by GTG-banding of the same metaphase spread. Confirmation of NOR localization was obtained by FISH analysis using human rRNA DNA as a probe.

SCIENTIFIC CONTRIBUTIONS OF THE MAP

Comparative mapping, in general, opens a door to the past since it allows us to recreate the evolutionary history of genomes and species. As more species are compared, more detailed information about evolutionary process will be elucidated. Although vole gene maps are not extensive at the time of this writing, they nevertheless provide valuable information for comparative analysis to study, in particular, the X chromosome evolution.

From comparative analyses of human and mouse X chromosomes, 10 conserved segments preserved for more than 100 million yr of independent evolution have been revealed (Blair and others 1994; DeBry and Seldin 1996; Dinulos and others 1996). Mapping the representative members of the 2 largest conserved segments in 5 species of voles has provided a basis for analyzing the behavior of these segments during evolution and for comparing these data with those of human, mouse, and rat. Xist, Pgk1, and Gla (belonging to the first largest segment) are linked in all species; and Hprt and G6pd (from the second segment) are also found together. Comparison of the gene order among the 5 species revealed some differences, although these were mainly rearrangements between the segments rather than within the blocks of homology (Nesterova and others 1998). It was shown by extensive comparisons of G-banded chromosomes at a high level of resolution that vole X chromosomes demonstrate greater homology to the human than to the mouse X chromosome, as is true for the rat (Millwood and others 1997).

Involving 5 species of voles in comparative analysis of the X chromosome together with mouse and human has demonstrated that the number of X chromosome rearrangements occurring during evolution is much greater than previously supposed. The evolution of some of the vole species is accompanied by inter- and intrasegment inversions of X chromosome material. Nevertheless, specific blocks of sequences on the X are clearly well conserved in all mammalian species studied to date. We were able to highlight 2 such conserved regions from our comparative data: the small block Pgk1-Xist and Hprt-G6pd. It is possible that most of the rearrangements on the X chromosome are permitted during evolution as long as they do not disrupt the most functionally critical regions (Nesterova and others 1998).

ANTICIPATED FUTURE CONTRIBUTIONS OF THE MAP

Mapping additional X-linked genes in the species described above and others will help to shed more light on the significance of segment conservation with respect to the possible role of X chromosome-specific sequences in dosage compensation. Further genetic mapping of the common vole genomes followed by comparative genetic analysis of these data could be helpful in revealing the regularities underlying the persistence of the synteny of genes. These revelations, in turn, will add to the understanding of chromosomal organization and genome evolution in mammals.

USES OF THE MAP AND ACCESSIBILITY

The adverse effect of voles on plant and animal breeding is widespread. Because voles are sources and carriers of many diseases hazardous to humans and domesticated animals (such as tularemia, leptospirosis, and plague), knowledge of vole genetics could help to restrict their harmful influence.

CONCLUSION

Comparative mapping of the X chromosome covers 5 mammalian species of gray voles at the time of this writing. Regional localization of 5 X-linked genes has been determined in all 5 species, and NORs have been located in 4. We anticipate that cytogenetic mapping of the vole using up-to-date methods will continue.

¹Abbreviations used in this paper: FISH, fluorescence in situ hybridization; G-banding, Giemsa banding; GTG, Giemsa banding with trypsin; NOR, nucleolus organizer region.

ACKNOWLEDGMENTS

We thank members of the X Inactivation Group and Laboratory of Biochemical Genetics for their assistance and helpful discussion. The research was supported by grants from INTAS (94-2877), Russian Foundation for Fundamental Research (97-04-49231), and Medical Research Council of Great Britain; and by fellowships from the Royal Society and Royal Society/NATO to T. B. N.

REFERENCES

Blair HJ, Reed V, Laval SF, Boyd Y. 1994. New insights into the man-mouse comparative map of the X chromosome. Genomics 19:215 220.

Cooper JEK, Hsu TC. 1972. The C-band and G-band patterns of Microtus agrestis chromosomes. Cytogenetics II :295-304.

DeBry RW, Seldin MF. 1996. Human/mouse homology relationships. Genomics 33: 337-351.

Dinulos MB, Bassi MT, Rugarli El, Chapman V, Ballabio A, Disteche CM. 1996. A new region of conservation is defined between human and mouse X chromosomes. Genomics 35:244 247.

Howell WM, Black DA. 1980. Controlled silver staining of nucleolus organizer region with a protective colloidal developer: A 1 step method. Experientia 36:1014-1015.

ISCN [International System for Human Cytogenetic Nomenclature]. 1981. High-resolution banding (1981). Cytogenet Cell Genet 31:5-23.

Jackson RK. 1997. Unusual laboratory rodent species: Research uses, care, and associated biohazards. ILAR J 38:13-21.

Malygin VM. 1973. Karyotypes of Microtus transcaspicus: The karyological method to check whether the M. arvalis group voles are in a species of their own. Zool J 52:791-794 (In Russian).

Malygin VM. 1983. Systematics of Common Voles. Moscow Russia: Nauka (In Russian).

Mazurok NA, lsaenko AA, Nesterova TB, Zakian SM. 1996a. High-resolution G-banding of chromosomes in the common vole Microtus arvalis (Rodentia, Arvicolidae). Hereditas 124:229-232.

Mazurok NA, Nesterova TB, Zakian SM. 1995. High-resolution G-banding of chromosomes in Microtus subarvalis (Rodentia, Arvicolidae). Hereditas 123:47-52.

Mazurok NA, Rubtsov NB, Nesterova TB, Zakian SM. 1994. High-resolution G-banding of chromosomes in Microtus kirgisorum (Muridae, Rodentia). Cytogenet Cell Genet 67:2(18-210.

Mazurok N A, Rubtsova NV, lsaenko AA, Nesterova TB, Zakian SM. 1996b. Comparative analysis of chromosomes in Microtus transcaspicus and Microtus subarvalis (Rodentia. Arvicolidae): High-resolution G-banding and localization of NORs. Hereditas 124:243-25(I.

Meyer MN, Golenishchev FN, Radjably SI, Sablina OV. 1996. Voles (Subgenus Microtus Schrank) of Russia and Adjacent Territories. St. Petersburg Russia: Proceedings of the Zoological Institute. Vol 232 (In Russian).

Meyer MN, Jordan M, Walknowska J. 1967. A karyosystematic study of the some Microtus species. Folia Biol (Polska) 16:251-264.

Meyer MN, Orlov VN, Jordan M, Walknowska J. 1971. A karyosystematic study of Microtus transcaspicus ilaeus. Folia Biol (Polska) 19:73-78.

Meyer MN, Orlov VN. Sholl ED. 1972. Sibling species in Microtus arvalis group (Rodentia, Cricetidae). Zool J 51:724-738 (in Russian).

Millwood IY, Bihoreau MT, Gauguier D, Hyne G, Levy ER, Kreutz R, Lathrop GM, Monaco AP. 1997. A gene-based genetic linkage and comparative map of the rat X chromosome. Genomics 40:253-261.

Nesterova TB, Duthie SM, Mazurok NA, lsaenko AA, Rubtsova NV, Zakian SM, Brockdorff N. 1998. Comparative mapping of X chromosomes in vole species of the genus Microtus. Chromosome Res 6:41-48.

Nesterova TB, Mazurok NA, Matveeva NM, Shilov AG, Yantsen El, Ginsburg EKh, Goss S J, Zakian SM. 1994. Demonstration of the X-linkage and order of the genes GALA, G6PD, HPRT and PGK in two vole species of the genus Microtus. Cytogenet Cell Genet 65:250-255.

Orlov VN, Malygin VM. 1969. Two forms of 46 chromosome Microtus arvalis Pallas. In: The Mammals (Evolution, Karyology, Systematics, Faunistics). Materials for the 2nd All-Union Symposium on Mammals. Novosibirsk Russia: Nauka. p 143-144 (In Russian).

Penny GD, Kay GF, Sheardown SA, Rastan S, Brockdorff N. 1996. Requirement for Xist in X chromosome inactivation. Nature 379:131-137.

Renaud P. 1938. La formule chromosomale chez sept especes de Muscardinidae et de Microtinae indigenes. Rev Suisse Zool 45:349-383 (In French).

Zakian SM, Kulbakina NA, Meyer MN, Semenova LA, Bochkarev MN, Radjabli SI, Serov Ok 1987. Non-random inactivation of the X-chromosome in interspecific hybrid voles. Genet Res 50:23-27.

Zakian SM, Nesterova TB, Cheryaukene OV, Bochkarev MN. 1991. Heterochromatin as a factor affecting the inactivation of the X-chromosome in interspecific hybrid voles (Microtidae, Rodentia). Genet Res 58:105 110.

FIGURE 1 Gene map of the East European vole (Microtus rossiaemeridionalis). Only chromosomes with mapped genes are shown.

FIGURE 2 Gene map of the Transcaspian vole (Microtus transcaspicus). Only chromosomes with mapped genes are shown.

FIGURE 3 Gene map of the Kirgizian vole (Microtus kirgisorum). Only chromosomes with mapped genes are shown.

FIGURE 4 Gene map of the common vole (Microtus arvalis). (a) obscurus form; (b) arvalis form. Only chromosomes with mapped genes are shown.

FIGURE 5 Gene map of the X chromosome of the field vole (Microtus agreslis).