GENOMICS14, 302-308 (1992)

Mapping Human X-Linked Genes in the Phalangerid Marsupial

Trichosurus vulpecula LYNNE M. McKAY, JACLYNM. WATSON, AND JENNIFERA. MARSHALLGRAVES Department of Genetics and Human Variation, LaTrobe University, Bundoora, Vic 3083, Australia Received December2, 1991

We mapped 1 5 human X-chromosome markers in the c o m m o n b r u s h - t a i l e d p o s s u m , Trichosurus vulpecula (Kerr), w h i c h r e p r e s e n t s t h e A u s t r a l i a n m a r s u p i a l f a m i l y P h a l a n g e r i d a e . In situ h y b r i d i z a t i o n w a s u s e d to loc a l i z e h i g h l y c o n s e r v e d h u m a n X - l i n k e d g e n e s to c h r o m o s o m e s o f T. vulpecula d i p l o i d l i n e s . T e n g e n e s located on the long arm of the human X (human Xq genes) a l l m a p p e d to t h e p o s s u m X c h r o m o s o m e . H o w e v e r , a l l f i v e g e n e s l o c a t e d o n t h e s h o r t a r m o f t h e h u m a n X (hum a n Xp g e n e s ) m a p p e d to a u t o s o m e s . T h e s e f i n d i n g s confirm our previous work, which showed that the X chromosome in macropodid and dasyurid marsupials bears all the human Xq genes but none of the human Xp g e n e s s t u d i e d . T h i s s u g g e s t s t h a t t h e m a r s u p i a l X is h i g h l y c o n s e r v e d , b u t its g e n e c o n t e n t r e f l e c t s t h a t o f o n l y p a r t o f t h e e u t h e r i a n X, a r e s u l t c o n s i s t e n t w i t h o u r h y p o t h e s i s t h a t a n a u t o s o m a l r e g i o n w a s a d d e d to t h e X e a r l y i n e u t h e r i a n d i v e r g e n c e . © 1992 Academic Press, Inc.

INTRODUCTION Comparisons of gene arrangements in different species of eutherian (incorrectly called "placental") mammals have revealed that large chromosome regions are conserved even between different orders (summarized by O'Brien and Graves, 1991). By far the most extensive conservation of gene groupings is reported for the eutherian X chromosome, which is largely conserved in size (approximately 5% of the haploid genome) and completely conserved in gene content, as predicted by Ohno (1967). Marsupial mammals (Infraclass Metatheria) diverged from eutherians at least 120 million years before present (MYrBP), so a comparison of genes on the X in the two infraclasses could provide information about the gene content of the X in a common ancestor and about the course of evolution of mammalian sex chromosomes. Marsupial karyotypes are highly conserved (Rofe and Hayman, 1985). The basic marsupial X is smaller than that of eutherians, constituting about 3% rather than 5% of haploid genome (Hayman et al., 1982; Young et al., 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

1982). Early gene mapping using family studies indicated that the X of several kangaroo species (Family Macropodidae) shared one or more of the human Xlinked genes PGK, G6PD, and G L A (Richardson et al., 1971; Cooper et al., 1971, 1983; summarized by Graves, 1990; gene names and locus symbols in Table 1). Some of these genes were also found to be X-linked in a species of dasyurid marsupial (small carnivorous marsupials of the Family Dasyuridae) (Cooper et al., 1983). This conservation was confirmed and extended by early somatic cell genetic studies, using hybrids between H P R T - d e f i c i e n t rodent cell lines and cells from a variety of macropodid and dasyurid marsupials. These studies confirmed that PGK, G6PD, and G L A were borne on the X in all species, and added H P R T t o the conserved suite of genes (Graves et al., 1979; Donald and Hope, 1981; Dawson and Graves, 1984, 1986; Dobrovic and Graves, 1986). Later work using Southern blot analysis of rodent-marsupial cell hybrids probed by human cDNA sequences assigned to the marsupial X an additional nine genes, borne on the long arm of the human X (here referred to as human Xq genes), and in situ hybridization confirmed this and provided a regional assignment (Spencer et al., 1991a,b). This conservation of the macropodid and dasyurid X evidently did not extend to genes located on the short arm of the human X (here referred to as human Xp genes). Several cell hybrids with an intact marsupial X were found not to express the marsupial form of S T S (Dawson and Graves, 1986). Southern analysis of these cell hybrids excluded a further six human Xp genes from the X in two macropodid and one dasyurid marsupial species, and in situ hybridization localized several of these in two major clusters on autosomes in a macropodid and a dasyurid species (Sinclair et al., 1987, 1988; Spencer et al., 1991b; Watson et al., 1992). Since dasyurid and macropodid marsupials diverged from each other 45-50 MYrBP (Archer, 1984), this smaller conserved X may be shared by all marsupials. The shared autosomal clusters may represent arrangements of genes in the common marsupial ancestor. We here report gene mapping studies in the brushtailed possum (Trichosurus vulpecula), which is common

302

303

MARSUPIAL SEX CHROMOSOME EVOLUTION

TABLE I Locus name

Symbol

Location in human

Insert/vector

Source and Reference

Long-arm probes Cell cycle G1 phase defect

CCG1

Xqll-ql3

5.3 kb human cDNA in pUC118 1.1 kb red kangaroo genomic DNA in pBR322 1.9 kb human cDNA in pUC9

Phosphoglycerate kinase 1

PGK1

Xql3

Proteolipid protein

PLP

Xq21.3-q22

Coagulation factor IX

F9

Xq26.3-q27.1

1.5 kb human cDNA in pGEM

Coagulation factor XIIIc

F8

Xq28

L1 adhesion molecule

LICAM

Xq28

0.9 kb human cDNA in pUC12 6.6 kb mouse cDNA in pB1

Red cone pigment

RCP

Xq28

1.2 kb human cDNA in pUC18

Glucose-6-phosphate dehydrogenase

G6PD

Xq28

2.5 kb human cDNA in pUC18

Unknown unique expressed DNA sequence

GDX

Xq28

2.0 kb human cDNA in pUC18

Unknown unique expressed DNA sequence

P3

Xq28

2.3 kb human cDNA in pUC18

T. Nishimoto, Kyushi University, Japan (Sekiguchi et al., 1988) D. Cooper, Macquarie University, Australia (Van Daal et al., 1989) A. Naismith, Hospital for Sick Children, Toronto, Canada (Naismith et al., 1985) K. H. Choo, Royal Children's Hospital, Melbourne, Australia (Choo et al., 1982) J. Gitschier, San Francisco, California (Gitschier et al., 1985) J. Demergeoct, Centre D'Immunologie, Marseille, France (Lau et al., 1989) J. Nathans, Howard Hughes Medical Institute, Research Laboratories, Baltimore, Maryland (Nathans et al., 1986) D. Toniolo, Instituto de Genetica, Biochemica al Eucluzionistica, Pavia, Italy (Persico et al., 1986) D. Toniolo, Instituto de Genetica, Biochemica al Eucluzionistica, Pavia, Italy (Toniolo et al., 1988) D. Toniolo, Instituto de Genetica, Biochemica al Eucluzionistica, Pavia, Italy (Alcalay and Toniolo, 1988)

Short arm probes Amelogenin

AMG

Xp22.1-p22.31

630 kb human cDNA in pGEM-1

Zinc finger protein Y-linked

ZFY

Xp22.1-p21.3 a

1.3 kb human cDNA in pUC13

DNA polymerase a

POLA

Xp22.1-p21.3

2.9 kb human cDNA in pCD

Tissue inhibitor of metalloproteinases

TIMP

Xp11.3-p11.23

0.8 kb human cDNA in p91023(B)

Monoamine oxidase

MAOA

Xpll.3-p11.23

2.0 kb human cDNA in pUC19

E. Lau, University of Southern California, Los Angeles (Lau et al., 1989) D. Page, Whitehead Institute, Cambridge, Massachusetts (Page et al., 1987) T. Wang, Stanford University, California (Wong et al., 1988) S. Clark, Genetics Institute, Cambridge, Massachusetts (Heubner et al., 1986; Willard et al., 1989) J. Powell Harrow, UK (Powell et al., 1989)

a Position of X-linked homolog of ZFY.

in southern Australia, and frequently used as a model for studies of marsupial physiology and reproduction. This species represents a third family of Australian marsupials, the Phalangeridae, which diverged from the Macropodidae about 40 MYrBP and from the Dasyuridae 50 MYrBP (Archer, 1984). In this species, the expression of P G K isozymes showed a population distribution consistent with X linkage and paternal inactivation (VandeBerg et al., 1979). Here we report the use of in situ hybridization to localize P G K and 14 other human X-linked markers in the possum. We found that, as for macropodid and dasyurid marsupials, human Xq markers mapped to the possum

X, but human Xp markers were autosomal, suggesting that this was the ancestral arrangement in marsupials. MATERIALS AND METHODS Cells and cell culture. Male and female brush-tailed possums ( Trichosurus vulpecula, Kerr, marsupial Family Phalangeridae) were

trapped at La Trobe University under Department of Conservation, Forests and Lands Permit No. 86/27. Ear-punch biopsies were ohtained from anesthetized animals, and primary cultures were obtained either by a plasma clot method or by collagenase dissociation. Cells grew readily, and cultures remained diploid over many months. Cells were grown in Dulbecco modified Eagle's medium (Flow, Australia) supplemented with 10% fetal calf serum (GIBCO) as well as

304

MCKAY, WATSON, AND GRAVES

:.

RCP

:: ...

::

:

1..

:

%" G6PD

• ee

..*

•e

..:ii: ........

:::: :::::.'-"

: :: . . :

:..:.'.

::.: i.ii:

::ii .i.: .ii

CCG1

::

.

. . : .*e.

ee.eeoe

• •

*.

.e.

: : 00

.0".:•

:e.

o• •

.e

...

.:.:

ee.

GDX

::

• ....

ii:iii o•

z .