Isolation and regional localization of the murine homeobox-containing gene Hox-3.3 to mouse chromosome region 15E

5,76-83

GENOMICS

(1989)

Isolation and Regional Localization of the Murine HomeoboxContaining Gene Hox-3.3 to Mouse Chromosome Region 15E KLAUS SCHUGHART,* Departments

DIMITRINA PRAVTCHEVA,* MARILYN S. NEWMAN,* ZHILING JIANG,* AND FRANK H. RumLE*,t

of *Biology and tHuman Received

Genetics, Yale University, New Haven, Connecticut

December

7, 1988;

INTRODUCTION

The homeobox is a highly conserved 180-bp DNA sequence present in several genes of Drosophila melanogaster affecting embryonic development of the fruitfly (reviewed by Scott and O’Farrell, 1986; Gehring and Hiromi, 1986). Homeobox-containing genes have also been detected in many other species, including mouse and human (McGinnis et al., 1984a). The high degree of sequence conservation among different species suggests that homeobox genes may play an important role during the embryonic development of vertebrates. Today, about 20 homeobox genes, most of which are organized in gene clusters, have been described in the mouse genome. The Hox-1 cluster on chromosome 6 contains at least 7 genes (McGinnis et aZ.,198413,Colberg-Poley et al., 1985; Bucan et al., 1986; Duboule et al., 1986; Rubin et al., 1986, 1987; Baron et al., 1987; Odenwald et al., 1987), and 7 homeobox genes have been identified in the Hox-2 cluster on mouse chromosome 11 (Jackson et al., 1985; Hauser et al., 1985; Hart et al., 1987; Rabin et al., 1985; Joyner et al., 1985; Miincke et al., 1986; Do and Lonai, 1988; Graham et aZ., 1988, Bogarad et al., 1989). Two homeobox genes, Hox-3.1 and -3.2, have been localized on chromosome data

from Data

this article have been deposited with Libraries under Accession No. 503074.

osss-7543/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

revised

February

06511

7, 1989

15 (Awgulewitsch et al., 1986; Rabin et al., 1986; Breier et al., 1988) and two homeobox gene loci map to chromosome 2 (Featherstone et al., 1988, Pravtcheva et al., personal communication). The En-l and En-2 homeobox genes have been localized as single gene loci on chromosomes 1 and 5, respectively (Joyner and Martin, 1987). The chromosomal locations of the Hox-6.1 and -6.2 homeobox genes (Sharpe et al, 1988) are discussed in this paper. Here, we report the isolation of the murine homeobox gene, Hox-3.3, and its localization to mouse chromosome 15. The mouse Hox-3 gene cluster may contain at least four homeobox genesorganized in a way similar to that of the genes in the Hox-1 and Hox-2 cluster.

A murine homeobox-containing cDNA clone has been isolated from an adult spinal cord library. Using in situ hybridization and somatic cell genetics techniques, the newly isolated homeobox gene has been mapped to mouse chromosome region 16E. Because of its chromosomal location, we called this gene locus Hox-3.3. Nucleotide sequence analysis revealed that the Hox-3.3 gene represents the murine cognate of the human homeobox gene c8. The presumptive organization of the murine Hex-3 homeobox gene cluster is 0 IQSB Academic Press, IIIC. discussed.

Sequence EMBL/GenBank

LISA W. HUNIHAN,*

MATERIAL

AND

METHODS

Isolation and Sequencing of cDNA Clones Approximately 4 X lo5 XgtlO recombinant phage clones representing a cDNA library from the spinal cord of adult mice were screened with a 353-bp genomic fragment of the murine Hoz-2.2 gene (Schughart et al., 1988) containing the Hox-2.2 homeobox. Overlapping fragments of the isolated cDNA clone cl6 were subcloned into Ml3 vectors (Maniatis et al., 1982) and the sequence from both strands of the l.O-kb cDNA insert was determined by the dideoxy sequencing method (Sanger et al., 1977) using Sequenase (USB Inc., Cleveland, OH). Sequence analyses were performed with computer programs provided by DNAstar (Madison, WI) and the University of Wisconsin Genetics Computer Group (Devereux et al., 1984). Chromosomal Mapping Formation and growth conditions of mouse hamster hybrid cell lines have been described elsewhere (Pravtcheva and Ruddle, 1983). Chromosome contents of hybrid cell lines were determined by karyotype and isozyme analysis (Pravtcheva and Ruddle, 1983). DNA isolated from hybrid cell lines was digested with HindIII and analyzed by Southern blot hybridizations using

the

76

MURINE

HOMEOBOX

the purified l.O-kb insert from clone cl6 as a probe (Maniatis et al., 1982). Metaphase chromosomes were prepared from karyotypically normal primary mouse embryo fibroblasts and from a primary cell culture of karyotypically normal liver cells established from newborn mice. [3H]dATP-, [3H]dCTP-, and [3H]TTPlabeled nucleotides (Amersham, Arlington Heights, IL) were used to label the l.O-kb insert of clone cl6 to a specific activity of approximately 2-4 X lOa cpm/pg by the random primer method (Feinberg and Vogelstein, 1983). In situ hybridizations with probe concentrations of approximately 2-5 X 10’ cpm/ml were performed as described by Rabin et al. (1984). RESULTS

Isolation and Structure Gene Hox-3.3

AND

DISCUSSION

of the Murine

Homeobox

Most murine homeobox-containing genes have been found to be expressed in the spinal cord of adult mice. Therefore, we prepared a cDNA library from mRNA of the spinal cord of adult mice in order to isolate homeobox-containing cDNA clones. When the library was screened with a Hox-2.2-specific probe, several clones containing the Hox-2.2 homeobox were isolated and analyzed in detail (Schughart et aZ., 1988a). In addition, another cDNA clone (~16) representing the transcript from a different homeobox gene was isolated. The structure of clone cl6 was further analyzed. We have demonstrated that this newly isolated homeobox gene is located on mouse chromosome 15, and in ref-

GENE

77

Hor-3.3

erence to the suggested nomenclature system for homeobox genes (Martin et al., 1987), we have called this gene Hox-3.3. Southern blot analysis of mouse genomic DNA identified Hox-3.3-specific restriction fragments of 6.4, 5.2,4.5, and 3.0 kb after digestion with restriction enzymes HindIII, EcoRI, BglII, and BamHI, respectively (data not shown). The digestion of DNA with BumHI also showed two additional weak bands of 2.2 and 2.0 kb. These results indicate that only a single Hex-3.3 gene locus is present in the mouse genome. Sequence analysis of clone cl6 (Fig. 1) revealed that it represents an incomplete cDNA clone with an open reading frame of 309 bp and a 3’ untranslated region of 667 bp. The identified open reading frame begins 8 amino acids upstream of the Hox-3.3 homeodomain and ends at a stop codon 34 amino acids downstream of the homeodomain. The nucleotide sequence of clone cl6 is almost identical to sequences described for two other murine homeobox-containing cDNAs isolated by Sharpe et al. (1988) and Kongsuwan et al. (1938). Sharpe et al. called the homeobox gene isolated in their laboratory Hox6.1. The following findings led us to the conclusion that our Hox-3.3 cDNA clone, the Hox-6.1 cDNA clone, and the cDNA isolated by Kongsuwan et al. (1988) represent different cDNA clones derived from the same murine homeobox gene. The Hox-3.3 and Hox-6.1 homeobox genes exhibit the same restriction fragment patterns after digestion of genomic DNA with restriction enzymes HindIII, EcoRI, and BanHI. In regions that are contained in both cDNA clones (clone cl6

AGT GGG GTC GGT TAC GGA GCG GAC CGG AGG CGC GGC CGC CAG ATC TAC TCT CGG TAC CAG ACC CTG GAA CTG GAG Ser Gly Val Gly Tyr Gly Ala Asp Arg Arg Arg Gly Arg Gln Ile Tyr Ser Arg Tyr Gin Thr Leu Glu Leu Glu

75 25

AAG GAA TTT CAC TTC AAC CGC TAC CTA ACT CGG CGC CGG CGC ATC GAG ATC GCC AAT GCT CTG TGC CTG ACC GAG Lys Glu Phe His Phe Am Arg Tyr Leu Thr Arg Arg Arg Arg Ile Glu Ile Ala Asn Ala Leu Cys Leu Thr Glu

150 50

GCG CGA CAG ATC AAA ATC TGG TTC CAG AAC CGC CGC ATG AAG TGG AAA AAA GAA TCT AAT CTC ACG TCC ACA CTC TCA Arg Gin Ile Lys Ile Trp Phe Gin Asn Arg Arg Met Lys Trp Lys Lys Glu Ser Asn Leu Thr Ser Thr Leu Ser

225 75

GGG GGT GGC GGA GGG GCA ACC GCC GAC AGC CTG GGA GGA AAii GAG GAA AAG CGA GAA GAG ACA GAA GAA GAG AAG Gly Gly Gly Gly Gly Ala Thr Ala Asp Ser -Leu Gly Gly Lys Glu Glu Lys Arg Glu Glu Thr Glu Glu Glu Lys

300 100

CAG AAA GAG TGACCAGGACTGTCCTTGCACCCCTCTCTTCTCCCTTGCTCATCCCAGCTCCCCGAATCACACACTCTGTATTTATCTCTGGCACAA Gin Lys Glu TRM

396 103

TGATGTGTTGTGACTTCCTAAAACAAAACAGGGAGTCAGACGTGGACCTAAAAGTCAGTGCACAACTCTTCACTCGTCT

49.5

CTTCCTCTAGCTCCCTCGCTAGCTTCTTCCTGGCTTGTCTGCAGGCCCCTTCCCTCGCCTAGGCCTAGGAGACCTGAACA

594

TGTCTCTCCAAGTGAGCCCTAGGCTCCCTCCCCACCCCCGACGCCCTTCACCCCCGCCCTCCGGCGCAGAGAGCTGGCTCCAGGGCCTGGGGCCTCCAC

693

TCGGGGGCCTCAGGGCCGGCCTGACAGCCGGAGGGCAGGAGGCCTCAGGAGGGCGCGCCTTGGCCCTGCAACAACCCCCAGGGCCTCTCCCCAGTCCCT

792

GCCCGGTCCCTCTGCCCCAGCAAATGCCCAGTCCAGGCAACGTTTCTG

801

TGTGAATATTTCTAGCTGTATTTGTGGTCTCTGTATTTATATTTATGTTTAGCACCATCAGTGTTCCTATCTCATTTAAAAAAGC

976

FIG. 1. Nucleotide 3.3 homeobox sequence of the human c8 protein

sequence of the isolated Hox-3.3 cDNA clone and conceptual translation of the largest open reading frame. The Hoxis boxed. The single amino acid residue in which the predicted Hor-3.3 protein differs from the amino acid sequence (51) is underlined. Differences from the cDNA sequence described by Sharpe et al. (50) are indicated.

78

SCHUGHART

contains 312 nucleotide residues of the 318 residues present in the Hox-6.1 cDNA clone), the nucleotide sequences of the Hox-3.3 and Hox-6.1 cDNA clones differ at only four positions. Between the nucleotide sequence of the cDNA clone isolated by Kongsuwan et al. and the Hox-3.3 cDNA clone, 16 differences have been noticed, 11 of which are present in the 3’ untranslated region. None of the differences between the nucleotide sequences of the three independently isolated cDNA clones results in a change of the predicted amino acid sequence of the Hex-3.3 homeobox gene product. The differences found might result from artifacts caused during reverse transcription of the mRNA templates, or the different cDNA clones might represent different allelic versions of the Hox-3.3 gene. Comparison of the nucleotide sequence of the Hox3.3 homeobox with other homeobox sequences revealed high, degrees of sequence conservation to the mouse Hex-1.1 homeobox (83.6%), the mouse Hox-2.2 homeobox (82.5%), the mouse Hox-1.2 homeobox (84.2%), the human c8 homeobox (96.2%), the frog XlHboxl homeobox. (80.9%), and the newt NvHboxl homeobox (84.2%; references see Fig. 4). The predicted amino acid sequence of the Hox-3.3 homeodomain is identical to the amino acid sequence of the human c8 and the Xenopus laevis XlHboxl homeodomain and highly similar (98.4%) to the newt NvHboxl homeodomain (Fig. 4). Furthermore, the predicted amino acid sequence of the open reading frame in clone cl6 (regions flanking the homeodomain included) is identical to that of the human c8 gene, with the exception of a leucine residue at position 86. In addition, the nucleotide sequence of the mouse Hox-3.3 clone cl6 is 88% similar to the sequence of the appropriate region in the human c8 homeobox gene. These findings suggest that the Hox3.3 gene represents the murine cognate of the human c8 homeobox gene. Also, within the 3’ untranslated region (3’ UTR) the Hox-3.3 gene exhibits 85% similarity to the 3’ UTR of the human c8 gene, indicating that these regions may have been conserved because they are biologically important. However, a possible functional role of the 3’ UTRs remains to be determined. The Mouse Hox-3.3 Locus Maps to Chromosome

15

To determine the chromosomal location of the mouse Hex-3.3 gene, we performed Southern blot analysis of DNA isolated from mouse-hamster hybrid cell lines and in situ hybridizations on mouse metaphase chromosomes. Southern blot hybridization yielded a HindIII fragment of 6.4 kb in DNA from the mouse genome that was clearly distinguishable from the homologous gene in hamster DNA (Fig. 2) Figure 2 and Table 1 show the analysis of 15 mouse-hamster hybrid cell lines

ET

AL.

FIG. 2. Southern blot analysis of mouse hamster hybrid cell lines. DNA isolated from hybrid cell lines was digested with Hind111 and hybridized to the Hex-3.3 cDNA probe. The bands in lanes labeled “CDl” (mouse genomic DNA) and “E36” (hamster genomic DNA) identify the mouse and hamster HOP3.3 homologs, respectively. The presence of a 6.4-kb fragment indicates the presence of the mouse Hor-3.3 gene. Mouse chromosomes that are present in each cell line are listed in Table 1.

containing different sets of mouse chromosomes. In all cell lines analyzed, the presence or absence of the Hox3.3 hybridization signal is concordant with the presence or absence of mouse chromosome 15. These findings demonstrate that Hox-3.3 is localized on mouse chromosome 15. Our results disagree with in situ hybridization data obtained by Sharpe et al. (1988), who mapped this gene to mouse chromosome 14. In our analysis of hybrid cell lines, however, we have detected 8 cell lines in which the presence or absence of Hox3.3 hybridization is discordant with the presence or absence of mouse chromosome 14 (Table 1). Therefore, we conclude that the mouse Hox-3.3 gene represented by cDNA clone cl6 and the cDNA clones isolated by Sharpe et al. (1988) and Kongsuwan et al. (1988) maps to mouse chromosome 15 and not to chromosome 14. According to the accepted nomenclature this gene locus should be called Hox-3.3. To regionally localize Hox-3.3 on mouse chromosome 15, we performed in situ hybridizations to metaphase chromosomes. In an analysis of 118 metaphases, 266 grains were scored, 13% of the grains were detected on chromosome 15 and only 2% of the grains were found on chromosome 14. These results confirmed our mapping data obtained with somatic cell hybrids. Of the grains detected on chromosome 15, 75% are localized over bands 15D-F with a peak in region 15E (Fig. 3). These observations indicate that the Hox-3.3 gene is located close to the region to which the murine Hox3.1 gene has been mapped previously by Rabin et al.

MURINE

TABLE Segregation

of Hex-3.3 Mouse

in Mouse-Hamster

Hybrids

Presence/absence of Hex-3.3

All

126/2

+ -

mc8

6*, 8*, 13, 15, 16, 17, X 1, x 2, 4, 7, 9, 12, 13, 19, x

mFEl1 ma106 R44

All autosomes, X 12, x 17, 18, unidentified

+ -

4B3/Az3 c17B

2, 7, 12, 13*,

2, 5, 6*, 8*, 10, 12, 13,

14, 15, 16, 17, 19, x m&19

-

chromosomes 15, 19 1, 2, 3, 4, 7, 9, 12, 15, 17*, 19, x* 1, 2, 3, 4, 5, 6, 7, 8**, 9, lo**, 12, 13, 15, 16, 17, 19, X

+ +

12G/1

5, 6*,

12, 13, 14, 16,

-

2ac2

17, 19, x 1, 2, 3, 4, 7, 8, 9, 10, 12, 13, 15, 16, 17, 18,

+

cl1

8*,

+

19, x mFE

2/1/l

1, 2, 3, 4, 6, 7, 8, 9, 10, 12, 13, 15, 17, 18, 19,

+

X 14*, 15 None

Ecm4 E36 (hamster) mFE 2/l/7’

+ -

1, 2, 3, 6, 7, 8, 9, 12, 13, 15, 17, 19, x

Presence

+

of chromosome/hybridization

Concordant Mouse chromosome

+/+

14

3

15

10

-/4 5

signal Discordant

Total

+/-

7 15

0

1

-/+ 7 0

Hon-3.3

79

Structure of the b4urin.e Hex-3 Homeobox Gene Cluster

+ +

CD1 (mouse) TuCE 12G/9

GENE

mapping data, these findings suggest that the murine Hox-3 cluster contains at least four homeobox genes (Hox-3.1, -3.2, -3.3, and -3.4; see Fig. 5) on chromosome 15.

1

chromosomes present

Ceil line

HOMEOBOX

Total 8 0

Note. List of mouse chromosomes present in different mouse hamster hybrid cell lines and summary of Southern blot analysis of DNAs from 15 different mouse-hamster hybrid cell lines. *, Rearranged chromosomes; **, chromosomes present in 15% of cells. ’ Data not shown.

(1986). Therefore, it is likely that the Hex-3.3 gene represents another member of the Hex-3 gene cluster on chromosome 15. Mapping analyses of genes in the human Hoz-3 gene cluster revealed similar results. Rabin et al. (1986) and Cannizzaro et al. (1987) showed that the human homolog of the mouse Hox-3.1 gene and the human c8 homeobox gene, which represents the human homolog of the murine Hox-3.3 gene, have been mapped to the same region on human chromosome 12. Sharpe et al. (1988) identified another homeobox downstream of the Hox-3.3 gene. Together with our

Our current knowledge about the structural organization of the murine Hox-1 and Hox-2 homeobox gene clusters and the sequence similarities between genes in these clusters prompted us to organize the Hox-3 homeobox genes in a presumptive Hox-3 cluster (shown in Fig. 5). Several genes in the murine Hox-1 and Hox-2 homeobox gene cluster have been identified and their sequence and structural organization within the gene clusters have been described (see Introduction). Comparisons of the homeodomains from individual genes reveal that pairs of genes with high sequence similarity can be found between the Hox-1 and Hox-2 clusters (Fig. 4). For some homeobox genes the entire open reading frame has been described. Comparisons of the amino acid sequences of the predicted proteins indicate that the Hox-2.1 and -1.3 genes (Fibi et al., 1988; Papalopulu et al., 1989), the Hox-2.3 and -1.1 genes (Schughart et al., unpublished), and the Hox-2.4 and -3.1 genes (Blatt et al., 1988) represent pairs of closely related homeobox genes sharing sequence similarities that extend to regions outside of the homeodomain. In addition, the relative order of related genes within each cluster is identical and the relative distances of related genes in the Hox-1 and Hox-2 clusters are very similar (Fig. 5; Do and Lonai, 1988; Graham et al., 1988). On the basis of these criteria, the genes from one cluster can be aligned with genes in the other cluster as illustrated in Fig. 5 (Hart et al., 1987; Do and Lonai, 1988, Graham et al., 1988; Duboule et aZ., 1989; Schughart et al., 198813). For example, the Hox-1.2 homeobox sequence shares the most identity with the Hox-2.2 homeobox and the distance of the Hox-1.2 homeobox to the neighboring Hox-1.1 and -1.3 genes in the Hox-1 cluster is similar to the spacing between the Hox-2.2, -2.1, and -2.3 homeobox genes in the Hox-2 cluster. The Hox-1.1 I-2.3 and Hox-1.31-2.1 homeobox genes also represent two pairs of related genes. Furthermore, the proximal-distal order of the Hox-2 genes (i.e., Hox-2.3, -2.2, -2.1) in the Hox-2 cluster is identical to the order of their cognate genes in the Hox-1 cluster (Hox-1.1, -1.2, -1.3). The same is true for the rest of the pairs of genes along the Hex-1 and Hex-2 cluster. Similarly, the Hox-3 genes can be aligned to genes from the Hex-1 and Hox-2 cluster as shown in Figs. 4 and 5. On the basis of the amino acid sequences of the homeodomain, the Hox-3.2 gene shares high sequence similarity with the Hex-1.7 and the Hex-2.5 genes (Fig.

80

SCHUGHART

ET

AL.

L oTYv??-F~T~t~T?~m

1. Y

I

FIG. 3. Chromosomal localization of the murine Hox-3.3 homeobox gene. Distribution of silver grains over mouse chromosomes after in situ hybridization with the Hex-3.3 probe are shown. Grain locations were recorded in reference to major bands on G-banded mouse metaphase chromosomes. The Hox-3.3 hybridization signal is localized on chromosome 15 in region 15D-F with a peak of grains over region 15E.

4 and Bogarad et al., 1989). Also, the homeodomain of the neighboring Hox-3.1 gene is similar to the Hox-2.4 homeodomain, and the Hox-3.3 homeodomain shows high similarity to the Hox-2.2 and -1.2 homeodomains. Furthermore, the distance between the Hox-3.3 and the Hox-3.4 genes (Sharpe et al., 1988) is similar to the distances separating Hox-2.2 and -2.1 or Hox-1.2 and -1.3. The sequence similarities and the similar spatial arrangement strongly suggest that the Hox-1.2, -2.2,

and -3.3 genes belong to the same group of related homeobox genes. These findings could indicate that the Hox-3 cluster and the Hox-1 and Hoc2 homeobox gene clusters may have evolved by duplications of a common precursor cluster and that the Hox-3 cluster may be organized in a way similar to that of the Hox-1 and Hox-2 clusters. Therefore, it may be possible to predict the most likely organization of the murine Hox-3 cluster (Fig. 5). We would also expect that additional hom-

References Antp

d

RKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKENK

Hox-l.lm Hex-3.2m

(33) (44) (6)

Hox-2.4m Hox-3. lm HOX-3.lh

-R------S-----------L--p----K---G-----"---------------N -RS-----S-----------L--p----K---G-----"---------------N -RT-----S-----------L--p----K----"s---G-----"---------------N

Hox-l.lm HOP2.3m

__-_--___-_________------------------------------------y------------T--T------------------------

Hox-1.2m Hox-2.2m Hox-3.3m c8 h XlHbOXl NvHboxl

GR---------------------------------N------------------------GR-------------------y-------------------------------------g-R----I-S--------------------------N-----------------------aN -R----I-S--------------------------N-----------------------6N -R----I-S--------------------------N-----------------------gN -R----I-S--------------------------N-a---------------------aN

(3.20) (1,6,29) (5) ------------------------H-

(25)

(20,26,30,35) (10) (20,30) (this work,26,50) (51) (9)

(46)

Hox-1.3m Hox-2.lm cpll h

(17.37) (21,22,27) (5)

Hox-l'.4m Hox-2.6m cp19 h

(13.43)

Hox-1.5m Box-2.lm

(34) (19,30)

Hox-1.6m

(x2,19) (5)

PNA”-TNF-TK-LT---------K----A--“---AS-Q-N-T-”----------Q--RE-

(2,28)

FIG. 4. Alignment of mouse and human borneodomain sequences. Amino acid sequences of the homeodomain from known murine (m) Hor-I, Hor-2, and Hor-3 genes and human (h) HOX-3 genes were used to group closely related genes. Also, alignments of the Hox-3.3 homeodomain to the closely related homeodomains of the XlHboxl (Xenopus) and the NuHboxl (newt) gene are shown. Dashes indicate amino acid residues identical to the Drosophila (d) Antp homeodomain.

MURINE 1.2

1.3

HOMEOBOX 1.4

GENE

I I 40

81

Hox-3.3 1.5

1.6

, I

I

I I

I 1

80

mouse Hox-1 CHR.6

mouse Hox-2 CHR.11

mouse Hox-3 CHR.15

3.1

I 10

I 20

es Iti

opll 7

I I

op19 d ‘I

30

40

50

human HOX-3 CHR.12

FIG. 5. Chromosomal organization of the Mouse Hex-1 and HOP2 homeobox gene clusters and predicted organization of the mouse Hox3 cluster. Vertical bars outline groups of closely related homeobox genes from different gene clusters. Question marks indicate that the precise location and orientation of some homeobox genes within the respective clusters have not yet been determined. The organization of the human Hex-3 gene cluster is shown for comparison. *The similarity of the Hex-2.5 homeodomain to the Hex-1.7 and Hox-3.2 homeodomains has been described by Bogarad et al. (4). For additional references see text and Fig. 4.

eobox genes paralogous to the genes in the Hox-1 and Hox-2 cluster which have not been described to date will be found in the murine Hox-3 gene cluster. Additional support for the presumptive structure of the murine Hox-3 cluster comes from data on human homeobox genes. Boncinelli et al. (1988) identified four genesin the human HOX-3 cluster (HOX-3.1, c8, cpll, and cp19) on human chromosome 12, one of which (~8) represents the homolog of the mouse Hox-3.3 gene. As illustrated in Fig. 5, the distances between mouse Hox3.3 and -3.4 and the human c8 and cpll homeobox genes are very similar. In addition, the human HOX3.1, c8, cpll, and cp19 homeobox genes show high similarities to four groups of related murine homeobox genes: the Hox-3.11-2.4 homeobox genes, the Hox-1.21 -2.2/-3.3 homeobox genes, the Hox-1.31-2.1 homeobox genes, and the Hox-1.41-2.6 homeobox genes, respectively (Fig. 4). Therefore, the mouseHox-3.4 gene might represent the murine cognate of the human cpll homeobox gene and one could expect to find another mouse homeobox gene that represents the murine homolog of the human cpl9 homeobox gene. These findings strongly indicate that our assumptions on the structural organization of the murine Hox-3 cluster are correct. In the future, it will be important to isolate additional genomic clones from the murine Hox-3 cluster which will allow us to determine whether the Hox-3.3 gene locus can be linked physically to the previously described Hox-3.1 and -3.2 genes and whether additional homeobox gene loci within the mouse Hex-3 cluster can be identified.

ACKNOWLEDGMENTS We thank many colleagues in our laboratory for helpful discussions and critical review of the manuscript. We thank Suzy Pafka for printing the figures. This work was supported hy National Institutes of Health Grant GM099966 and an anonymous gift to F.H.R. K.S. is supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (FRG).

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2.

BARON, A., FEATHERSTONE, M. S.,HILL, R. E., HALL, A., GALLIOT, B., AND DUBOULE, D. (1987). Hox-1.6: A mouse homeobox-containing gene member of the Hox-1 complex. EMBO J. 6: 2977-2986.

3.

BLA’~~, C., ABERDAM, D., SCHWARTZ, R., AND SACHS, L. (1988). DNA rearrangement of a homeobox gene in myeloid leukaemic cells. EMBO J. 7: 4283-4290.

4.

BOGARAD, HART, C., expression Dev. Biol.

5.

BONCINELLI, E., SOMMA, R., ACAMPORA, D., PANNESE, M., D’ESPOSITO, M., FAIELLA, A., AND SIMEONE, A. (1966). Organization of human homeobox genes. Hum. Reprod. 3: 880-886.

6.

BREIER, G., DRESSLER, G. R., AND GRUSS, P. (1988). Primary structure and developmental expression pattern of Hox 3.1, member of the murine Hox 3 homeobox gene cluster. EMBO J. 7: 1329-1336.

I.

L. D., UTSET, M. F., AWGULEWITSCH, A., MIKI, T., AND RUDDLE, F. H. (1989). The developmental pattern of a new murine homeo box gene: Hox-2.5. 133: 537-549.

BUCAN, M., YANG-FJZNG, T., COLBERG-POLEY, A. M., WOLGEMUTH, D. J., GUENET, J.-L., FRANCKE, U., AND LEHRACH, H. (1966). Genetic and cytogenetic localisation of the homeo

a

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