Proc. Null. Acud. Sci. USA
Vol. 86, pp. 7716-7720, October 1989
Biochemistry

kmphila z&homeobox genesJ

  (NK-1, NK-2, NK-3, and NK-4 DNA clones/chromosome locations of genes)

YONGSOK KIM AND MARSHALL NIRENBERG

Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Building 36. Room lC-06. Bethesda, MD 20892

Contributed by Marshall Nirenberg, July 6, 1989

ABSTRACT i 6&r Drosophila melanoEaster homeohox
genes were found by screening a genomic fiNA library with
oligodeoxynucleotides that correspond to a conserved amino
acid sequence that is part of the putative site of homeobox
proteins that recognizes nucleotide sequences in DNA. The
amino acid sequences of NK-2, NK-3, and NK-4 homeoboxes
are more closely related to one another (59-66% homology)
than they are to other Drosophila homeoboxes (28-54% ho-
mology), whereas the homeobox of NK-1 is most closely related,
in order of decreasing homology, to muscle segment homeobox,
zerkniillt-1, NK-3, and distal-less homeoboxes. Three of the
genes, NK-1, NK-3, and NK-4, comprise a cluster of homeobox
genes located in the 9331-5 region of the right arm of the third
chromosome, whereas the fourth homeobox gene, NK-2, is
located in the lCl-5 region of the X chromosomd

Homeobox genes encode DNA binding proteins that regulate
gene expression during development or in the adult (l-4). In
most cases, the similarity between different kinds of ho-
meobox proteins extends only over a segment of the protein
that consists of 60-61 amino acid residues, the homeo-
domain, which is thought to be the portion of the protein that
recognizes nucleotide sequences in DNA. Homeobox genes
are particularly well expressed in nervous system, and the
homeobox family of genes encodes the largest set of proteins
that regulate gene expression in the nervous system that has
been identified thus far (5-9).

In this report, we describe four newly discovered, related
Drosophila homeobox genes that were detected with oligo-
nucleotide probes corresponding to an amino acid sequence
that is thought to be part of the nucleotide sequence recog-
nition site of homeobox proteins.

METHODS AND MATERIALS

Oligodeoxynucleotide. An Applied Biosystems DNA syn-
thesizer 380B was used to synthesize oligodeoxynucleotides.
Oligonucleotides with the trityl groups attached were purified
by OPC column chromatography and trityl groups then were
iemoved as described by Applied Biosystems. [y"P]ATP
with a specific activity of 6000 Ci/mmol (1 Ci = 37 GBq) (New
England Nuclear) was used for phosphorylation of oligode-
oxynucleotides catalyzed by T4 polynucleotide kinase (10).

Detection and Cloning of Homeobox Genes. A Drosophila
melanogas~er genomic DNA library in Charon 4A (11) was
obtained from the American Type Culture Collection. Recom-
binant phage [48,000 plaque-forming units (pfu)] and 2 x 109
Escherichia coli KH802 cells were plated in Petri dishes (150
mm) at a concentration of 12,000 pfu per dish. Four nitrocel-
lulose replica filter plaque lifts were obtained from each Petri
dish, and each filter was hybridized with a different [32P]-
oligodeoxynucleotide preparation [16-64 oligodeoxynucle-
otide species per preparation; 1.5 x lo6 cpm/ml; 120-150

The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "adverfisement"
in accordance with 18 U.S.C. 61734 solely to indicate this fact.

fmol/ml (the sum of all species of oligodeoxynucleotides)] at
37oC overnight and washed with a solution containing tetra-
methylammonium chloride at 53oC or 50oC for 30 min for 17-
mers or 16-mers, respectively, as described by Wood ef a/.
(12).

DNA Sequencing.* Cloned genomic DNA fragments cleaved
by restriction enzymes were subcloned into Bluescript pKS+.
Both strands of the homeobox regions of the following DNA
fragments were sequenced by the dideoxynucleotide chain-
termination method (13) using Ml3 universal primers or spe-
cific oligodeoxynucleotide primers and Sequenase 2 (United
States Biochemical): NK-1, 1.4-kilobase (kb) EcoRl/Pst I
DNA fragment; NK-2, 1.2-kb EcoRl/Psf I DNA fragment;
NK-3, 0.7-kb Pst I DNA fragment; NK-4, 0.4-kb and 2.3-kb
upstream Hind111 DNA fragments. dlTP was used to reduce
compression of DNA bands.

Locations of Genes on Chromosomes. Salivary gland poly-
tene chromosomes were hybridized with EcoRl-cleaved ge-
nomic DNA fragments that contained the appropriate ho-
meobox region and had incorporated biotin-16 dUMP in place
of some dTMP residues as described (14). Detek l-HRP kits
(Enzo Biochemicals) and the protocol supplied by the man-
ufacturer were used.

RESULTS AND DISCUSSION

Detection of Homeobox Genes. The Drosophila genomic
DNA library of Maniatis et al. (11) in Charon 4A was
screened for recombinants corresponding to homeobox
genes with five [32P]oligodeoxynucleotide probe preparations
designed to hybridize to highly conserved homeobox nucle-
otide sequences. The oligonucleotide preparations were 16 OJ
17 nucleotides long and each consisted of multiple species of
oligodeoxynucleotides (described in the legend to Fig. 2).
Replica filters were prepared and each filter was hybridized
to a different [32P]oligodeoxynucleotide preparation. The
filters were washed under high-stringency conditions with a
solution that contained tetramethylammonium chloride,
which selectively binds to A*T base pairs and raises the
melting temperature (fm) of A-T base pairs to that of CC base
pairs (12). The t, of each [32P]oligodeoxynucleotide-DNA
duplex then was dependent on the nuniber of contiguous base
pairs formed but was not affected by the proportion of GC vs.
A*T base pairs (12). Consequently, all species of 17-mer
oligodeoxynucleotides hybridized to DNA were washed ~11
the same temperature (53oC) for the stringent wash, and ;`I
16-mers were washed at 50oC.

Of the 48,000 phage plaques that were screened, =X1()
clones were obtained that exhibited a positive autoradio-
graphic signal with one of the five [32P]oligonucleotide probe
preparations, and 7 recombinant clones were obtained that
gave positive signals with two or more probe preparations.
Many of the 200 clones that were detected with only one

*The sequences reported in this paper for NK-1 to NK-4 have been
deposited in the GenBank data base (accession nos. M273sy.
M27290, M27291, M27292, respectively).

7716


Biochemistry: Kim and Nirenberg

Proc. Natl. Acad. Sci. USA 86 (1989)

7717

probe were cloned, but they were not studied further. The 7
clones that were detected with two or more 32P-labeled
probes were characterized by restriction site analysis and the
nucleotide sequences of the homeobox regions of some of the
clones were determined by using unlabeled probes as se-
quencing primers. Five of the 7 clones were found to be
previously unknown homeobox genes. The 2 remaining
,lones correspond to known homeobox genes; 1 clone con-
..iins zerkntillt-1 (zen-1) and Zen-2 DNA (15), and the other
clone corresponds to either en or inv (16) (data not shown).

Characterization of Homeobox Genes. In Fig. 1 are shown
partial restriction site maps of the homeobox genes NK-1,
NK-2, NK-3, and NK-4, the locations of homeobox regions
within the DNA inserts, and the direction of transcription. The
approximate chain lengths of the cloned NK-1 and NK-2
genomic DNA fragments are 15.0 and 14.1 kb, respectively.
Three of the 7 clones detected with two or more probes
correspond to NK-3. Clone 6 is a 14.7-kb DNA fragment that
L-ontains the NK-3 homeobox sequence. Clones 3 and 9
contain similar or identical DNA inserts (14.6 kb) that overlap
clone 6 and contain both NK-3 and NK-4 homeobox se-
quences separated by 27.8 kb. The restriction site map of
NK-3 and NK-4 shown is derived from data obtained from
clones 6, 3, and 9. Subcloned EcoRI DNA fragments from
clone 3 were used to determine NK-3 and NK-4 nucleotide

   ATGGCACCAACATGTGCCGAMAATTCCAATTRATCGAAC -29;

CCGTGGTGATTGATTTCCGTTTTCCAATCCCCCAGGACATT~CA~TGTCTGTGAT~ -234

ATGGCCCTAGCCTGTTGACTTATGCAARAAGAGAGACACCC~~CTTATCGT~C~ -175

Characterization of NK-2. The nucleotide sequence of the
homeobox region of the NK-2 gene is shown in Fig. 3. The
deduced amino acid sequence before the homeobox contains
repetitive asparagine residues and a highly acidic region
consisting of 14 aspartyl or glutamyl residues in a 31-amino
acid segment (45% acidic amino acid residues). Twenty-five
percent of the amino acid residues before the homeobox are
glycine plus alanine. The carboxyl-terminal 30 amino acid
residues of NK-2 are rich in histidine (20%), proline (17%),
and glycine (17%). A 168-nucleotide 3'-untranslated region
also is shown.

Characterization of NK-3 and NK-4. The nucleotide se-
quence and deduced amino acid sequence of part of the NK-3
homeobox gene are shown in Fig. 4. The initial part of the
sequence consists of part of an exon that encodes 54 amino
acids (17% alanine, 19% serine and threonine, and 9%
asparagine), which is followed by a short, 11Pnucleotide
intron within the 26th codon before the homeobox. The
intron-exon structure of the NK-3 gene was confirmed by
sequencing NK-3 cDNA clones (K. Webber, Y.K., and
M.N., unpublished data). The initial portion of the second

sequences.

Partial Nucleotide Sequence of NK-1. The sequence of an
811~nucleotide portion of the NK-1 gene is shown in Fig. 2.
The first 198 nucleotides correspond to the 3' portion of an
i Itron. Another intron, 217 nucleotides long, was found
\<ithin the homeobox, between codons for homeobox amino
acid residues 44 (glutamine) and 45 (valine). The intron-exon
structure of the NK-1 gene was confirmed by sequencing
NK-1 cDNA clones (to be described elsewhere). Three other
Drosophila homeobox genes, labial (lab) (19,20), abdominal-
B (Abd-f3) (21), and distal-less (D/l) (22) [Brista (23)] have
introns at precisely the same location within the homeobox as
NK-1. The intron within the NK-1 homeobox contains a
nucleotide sequence for antennapedia (Antp) protein binding
(18) and one or two binding sites for zeste protein [the
onsensus nucleotide sequence for zeste is TGAGYG (Y,
pyrimidine) (17)].

The amino acid sequence of the initial portion of the first
NK-1 exon shown in Fig. 2 is highly acidic-i.e., 12 of the
first 26 amino acid residues shown are aspartyl or glutamyl
residues. Twenty-five percent of the amino acid residues
before the homeobox are glycine residues, which include 7
consecutive glycine residues, and 17% are serine or threonine
residues.

w R    R     R H        A
~ NK-1 (CLONE 2)
   P                 B
IPI      H , `1,      RH        b?I
1 I
P P 8b         I lY    I
         88 P              P   NK-2 (CLONE 51
               n B     P

`l NK-3 lCLONE6)

NK-3&NK-4 (R,~,<-J             NK-I         IW
(CLONE3) )---t------- -

NK-3 BNK-4
COMPOSITE

FIG. 1.  Partial restriction maps of NK-1, NK-2, NK-3, and NK-4
cloned genomic DNA fragments. Solid boxes represent homeoboxes
and are not drawn to scale. Arrows indicate direction of transcrip-
tion. B. BmzHI: H. Hir>dllI: P. Pst 1; R, I3oRI; (R), EcoRl cloning
\rte created by ligation of an EcoRl linker to genomic DNA.

v

ATCTCCTCTTCTTTTTTTTGTCTTGCAG CC CAG GAT TTG AAT GAC ATG GAT -
               Gln m Leu Asn m Met iAspl

CAG GAC GAT ATG TGT GAC GAT GGC AGC GAT ATC GAC GAT CCC AGC
Gln(Aspd Met Cys RspAsplGly Ser m Ile P,d Pro Ser

AGC GAG ACG GAC TCC AAA AAG GGA GGC AGT CGT AAT GGG GAT GGA

rq Thr Ala,Phe Thr Tyr Glu Gin Leu Val Ser Leu Glu As,, Lys

he Lys Thr Thr Arq Tyr Leu Ser Val cys Glu Arq Leu Asn Leu

,124
-42

-79
-2,

-34
-12

12
4

5:
19

102
34

                     I
CC CTC AGC TTG AGC CTG ACA GAG ACG CAG GTGAGCAATGATATATACT  151
  Leu Ser Leu Ser Leu Thr Glu Thr Gin                44

TATTGTTAAAGATTAAAATCCAGAGAAGTTATGTATATTTTGCAAQAGTTGGTATAA 214

                                         26;

328

          --"-----------
ATTTCTCATCATCCTTTTACAG GTT AAA ATT TGG TTC CAG AAC CGC 0.X 377
               Val Lys Ile Trp Phe Gin As" Arq Arg   53

I

CC MG TGG AAG AAG CAG AAC CCC GGC ATG GAT GTC AAC TCC CCC 421
Thr Lys Trp Lys Lys Gin As" Pro Gly Met Asp Val Asn Ser Pro   68

ACC ATC CCC CCG CCC GGC GGC GGC TCC TTC GGA CCG GG        460
Thr Ile Pro Pro Pro Gly Gly Gly Sex Phe Gly Pro Gly        81

FIG. 2.  Nucleotide sequence and deduced amino acid sequence of
the homeobox region of the NK-1 gene. Deoxynucleotide and amino
acid residues are numbered on the right; 1 corresponds to the first
deoxynucleotide or amino acid residue in the homeobox, which is
enclosed in a large box. The acidic amino acid region is indicated by
boxed Asp or Glu residues. Repetitive Gly residues before the
homeobox also are enclosed in a box. The 1st and 2nd boxed
nucleotide sequences in intron 2 are possible sites for binding of zeste
protein to DNA (17). The 3rd site, ANNNNCATTA, is an Antp
protein binding site (18). Arrowheads represent intron-exon junc-
tions. Nucleotide 12 in an NK-1 genomic DNA clone was C. whereas
the corresponding nucleotide residue found in an NK-1 cDNA clone
was T. All oligodeoxynucleotide probes are complementary to the
DNA strand shown: probe sequences, starting from the 5'-terminal
nucleotide residues are as follows: -, probe 121, 24 species of
17-mers, (-)TNTGRAACCA(T/A/G)TARAA; - -, probe 125, 48
species of l7-mers, (-)AACCA(T/G/A)ATYTTNACYTG; --. probe
126,64 species of 17-mers, (-)AAYTCYTTYTCNAGYTC; - -, probe
127. 64 species of 17-mers, (-)-C(T/G)RTlYTCRTTRAAYTC; . . . ,
probe 130, 16 species of 16-mers, (-)A(C/G)T(C/G,(C/G)TIT/
G)CTCCAGCTC. Y, pyrimidine; R, purine.


7718   Biochemistry: Kim and Nirenberg                  Proc. Natl. Acad. Sci. USA 86 (1989)

exon before the homeobox consists of 35% serine and 19%
proline residues. The 54 amino acid residues after the ho-
meobox are rich in alanyl and glycyl residues (22%) as well
as leucyl residues (11%).

In Fig. 5 is shown the nucleotide sequence of part of the
NK-4 gene. The initial part of the sequence consists of part of
an intron, which is followed by an exon that contains the
homeobox domain. The carboxyl-terminal region of the de-
duced NK-4 protein contains repetitive glutamine residues (M
or opa repeats and a CAX repeat in the corresponding DNA).
Locations of Genes on Chromosomes. The cytological lo-
cations of the NK-1, NK-2, NK-3, and NK-4 genes in
Drosophila third-instar larvae salivary gland polytene chro-
mosomes are shown in Fig. 6. Unexpectedly, NK-1, NK-3,
and NK-4 genes were found to reside in neighboring chro-
mosomal bands in the right arm of chromosome 3. The NK-3
and NK-4 genes reside at 93El-3, and the NK-1 gene resides
at 93E3-5. When two probes, one for NK-1 and one for
NK-3, were added to the same in situ hybridization reaction
mixture, two labeled chromosomal bands were obtained at
93El-5 that were separated only slightly. However, the NK-2
gene resides in the lCl-5 region of the X chromosome. In
Fig. 6B, the relative positions of the NK-3/NK-4 and NK-1
genes are shown correlated with the chromosomal bands in
the 93E region of chromosome 3 in Bridges' revised map of
chromosomal bands (24). These results show that NK-1,
NK-3, and NK-4 comprise a cluster of homeobox genes.
Either NK-1, NK-3, or NK-4 genes may be the same as
torso-like, a maternal effect gene that resides at 93E and is
one of the ensemble of genes that determine the anterior-
posterior pattern of the embryo (2). The torso-like gene and
four other genes are required for the formation of both the
anterior and posterior terminal, unsegmented portions of the
embryo (the acron and telson) (2).

Another candidate is paired gene 9, which is thought to
contain repetitive alternating codons for histidine and proline
termed a paired repeat [also found in paired (25) and bicoid

KG GCC CRT KC CTIA CAC AAC ARC RAT AAT AAT ACG RCA AAC AAC -160
Thr Ala HIS Ala Leu "1sl4s.n Rsn Asr hsn ASK Thr Thr Asn Asn -54

RAT AAC CAC AK CTG RAG GCC GAG GGG ATC AAC GGA GCA GGC AGT -115
m HIS Ser Leu Lys hla Glu Giy Ile Asn Gly Ala Gly Ser  -39

GGT CAC GAC GRT AK CTC AAC GAA GAT GGC ATC GAG GAG GAT ATC  -70
Gly HIS iAsp Ser Le'x ~sn m Gly Ile klu Glu Asd Ile  -24

GAC GAC GTG GAC GRC GCC GAC GSC XT GGC GGC GGG GAT GCA RAT
As s
7 "al 1-d Ala m i;y Ser Gly Gly G1y vispl Ala As"
BamH                 -1 +1

GGA TCC GAC GGT CTG CCR ART RRG AM CGG ARG CGR CGA GTC CTG
Gly Serm Gly Le" Fro hsn Lys Lys Arg iys Arg Arq Val Leu

TTC ACC AAG GCG CR.4 ACR TAT GA; CTG GM CGT CGG TTT CGA CEA
Phe Thr Lys Ala Gin Thr Tyr Glu Le.2 Slii Rrg Rrg Fhe Arg Gln

CAA CGT TRC TTG AGT GCC 'CCG GAA CGC GAG CAC CTG GCC AGT TTG
Gin Arg Tyr Leu Ser Ala Frc Glu Arq Gli; HIS Leu Ala Ser Le"

ATC CGC CTG ACG CCG ACC CAG GIG AAG ATC TGG TTT CAA AAC CAT
Ile Arq Leu Thr Pr3 Tnr Gin Val Lys Ile Trp Fhe Gin As" tils

CGC TAC AAG ACG AA: CG:: GCG CAA AAC GAG AAG GGC TAC GAG GGT
&rg Tyr Lys Ttr Lys AIM A.la Gln As" Glu Lys Gly Tyr Glu Gly

CAT CCT GGT CTA CTG CAC GGC CAT GCC ACC CAT CCG CAT CAC Ccc  246
IHis Pro] Giy L~'J Leu a Gly a Ala Thr kis Pro His His Prd   82

AGT GCC CTG XA ?CG CCC G-C GGG TAG CCGYTCCRGTTCTGGTGAGGAAC
Ser Ala Leu m Ser m 'Jai Gly '**

GGAAAGeCCTGCTTGGGCGATAGT;CCF.ARCTGGGAGCCGACTGCG?CTCCGTGTCATC

AGCCACCGCCACCGCCATGCAGAATGeCSC`CCGC~CATCACTTGGTTGCCCTAAATGGAG
           Pst I
CGGCCGCCTATCAACATGCCGUUZ

-25
-9

21
7

6 6.
22

111
37

156
52

201
67

291
90

341

400

440

FIG. 3.  Nucleotide sequence and deduced amino acid sequence
of the homeobox region of NK-2 genomic DNA. The homeobox
domain is enclosed in a large box. Repetitive Asn residues are
enclosed in a box. The acidic amino acids enclosed in boxes before
the homeodomain comprise an acidic region of NK-2 protein.

(25) genes] and a homeobox, which resides at 93El-2 and was
cloned by Frigerio et al. (25). Elsewhere, we will show that
NK-1 contains a paired repeat (unpublished data): however,
it is not known whether NK-1 is the same as paired gene 9.
It should be noted that binding sites for polycomb protein
have been detected at 93El-4 (27). One of several candidates
for the NK-2 gene is twisted, discovered by Demerec et al.
(28), which is located between lC-5 and 2C-10. The abdo-
mens of adult TV' mutants, viewed from behind. are rotated
-30oC clockwise. However, further work is needed for the
identification of NK-1, NK-2, NK-3, and NK-4 genes.
Homeobox Homology. The deduced amino acid sequences
of the homeobox domains of NK-1, NK-2, NK-3, and NK-4
are shown in Fig. 7 and are compared with the 23 Drosophila
homeobox sequences that have been reported thus far. NK-2,
NK-3, and NK-4 homeoboxes are more closely related to one
another (59-66% homology) than they are to other Droso-
phila homeoboxes. The maximum homology to a previously
reported homeobox is to muscle segment homeobox (msh)
(29) (54% homology). In order of decreasing homology, the
homeobox of NK-1 is most closely related to msh, Zen-l,
NK-3 and Dll homeoboxes. NK-1, lab, Dll, and Abd-B genes
may have originated from a common precursor because each
gene contains an intron between the codons for the 44th and
45th homeobox amino acid residues. It has been suggested
that homeobox proteins bind to DNA via a helix-turn-helix
motif in the homeodomain and that amino acid residues 42,
43, and 47 in the third a-helix of the homeodomain interact
with nucleotide residues in the major groove of DNA and
determine, at least in part, the nucleotide sequence recog-
nized (29-31). Since NK-1, lab, Abd-B, and Dll genes each
contain an intron between the codons for the 44th and 45th
homeobox amino acid residues, the part of each gene that is
thought to encode the DNA recognition site of the corre-

Pst I
'C;G CAG TRT TAT GCG GCG Xc; ATG GA: AAC AAT RAC CAC CAT CAC -31r
LCJ Gin Tyr Tyr Ala Ala A;a ME: Asp Asn Asr. As" H15 H2s His -hi

CA; GCA ACG GGC ACA TCS AAC XC AGT GCC KC GAC TAC ATG CAC -z7:
Gin Ala Thr Gly Thr Ser ARC Ser SPI A13 Ala Asp Tyr Met Gin  -5:
           BornHI
CGC A.&A TTG KC TAT TX GGA TCC AC: CT: GCT XT ZCT TTG GAC -::;
Arq Lys Le" Ala Tyr Phe G1j Ser Thr Le" Ala Ala Pril re" Asp

AT; AGA cGC TGC AC:: AGZ AK SAT TCC  v
                      G GTA.AGTAACXCA:GAAAT`-A -I'-
Yet Arq Arq Cys Thr Ser Asr As? Ser A                 -2t

ACGCCATTCAGGcTcTAATIZGACTCTG~~AAGAAC^CCCT?TTGT -::f
                     v
ATRGGA_GTA~GCTAACTTT_GGTAAT~~~C'~~T~~*'~A~ AC TGC SAC TCA 'C:A -^'
                      sp Cys Asi; Ser Fro -:

CCG CCA TTG AC, AGT 'ICC CC. TCC GAG TCG CC: STA TCZ CAC GAC -!"
Prc Fro Leu Se: Ser Ser Pro Se: G1> Ser Fro Leu Ser 41s Asp   --


                      CGG TCG CGT GCC GCC TTC AGC   ^~
                                           -
                    Arq Ser Arq Ala A:a Fhe Ser,   '

AC GCC CRS G-C TTC GAG TT; GAC, CGC CG: 'XT XC CM CAG  ' 7:
                                       ':GCl
           Slu Lerl Glu Arg Arg Phe Ala Sin Gin Arg   ;i


AC TTG TCC GGT CCG GAA ZGC AX GAG RTG GCC AK RGC CTG CGC '3;
  !.eu Ser Gly Frs GIL Arg Ser Glu Me: Ala Lys SPL Leti Arq   '

             AAG ATS TGG TTC ZM AAC CGZ CGZ TAC lL-
  Thr Glu Thr Gin b'ril Lys ile :rp Phe Sin Asn Arq Arq Tyr   c


AA; AC': AAG            CA: CAC GAG XC XC CT TTG ;
                      Gin 91s G11: Ala Ala Leu ie"   
GG: GCC AGC AAG AGG GTT CCC GTC CAA GTC TT; GTG CGA GAG GAT 2::
Gly Ala Ser Lys Arq "al Prc '/a! Gin "al Leu "al Arg Glu ASP
GSC AX ACC ACC TAC GCT CAC ATG GCT GCT CCC GGT GCT GGA CAC ';I
G;y Ser Thr Thr Tyr Ala H15 Yet Aid Ala Pro Gly Ala Gly ,416   "
                       Pst !
SGC CTC GAT CCC GCC CTG ATC AAC ATC TPC CGC CAT i`AG CTG LAG ;;.;
Giy Le" Asp Pro Ala Leu Ile As" Ile Tyr Arq His GA" 'eu Gin

FIG. 4. Nucleotide sequence and deduced amino acid sequence
of the homeobox region of the NK-3 gene. The homeobox is enclosed
in a box. Arrowheads represent exon-intron junctions.


I -

Biochemistry: Kim and Nirenberg                 Proc. Natl. Acad. Sci. USA 86 (1989)   7719

spending protein is interrupted by an intron. Further work is
needed to determine whether the specificity of DNA recog-
nition by NK-1 protein is altered by alternative splicing.
Amino acid replacements that alter the 42nd or 43rd amino
acid residues in the homeobox are of special interest since they
may determine part of the nucleotide sequence that is recog-
nized by the homeobox protein. The 42nd homeobox amino
:Lcid residues of NK-2 and NK-4 are proline and alanine,
I espectively. The unspliced form of Saccharomyces cerevi-
:,,ue mating-type factor a-l has proline at this site (29); how-
ever, neither proline nor alanine has been found at this site in
any metazoan homeobox protein, A proline residue would not
be expected to be part of an a-helix, unlike alanine or glutamic
acid residues, which promote a-helix formation. The 43rd
homeobox amino acid residue of NK-1, NK-2, NK-3, and
NK-4 is threonine; however, the only other homeobox pro-
teins that contain threonine at this site are msh, Dll, lab, and
ro in Drosophila and Hox 1.6 and Hox 7.1 in the mouse.
The amino acid sequences of most or all of these homeobox
domains share other unusual features [for example, see alanine
( ilth amino acid residue), lysine or arginine (19th residue),
glutamic acid (30th residue), tyrosine (54th residue), and serine
or threonine (56th residue)]. The presence of the same or
similar unusual amino acid replacements in most or all ofthese
homeodomains provides additional evidence that the newly
discovered homeobox genes are related to one another.
The combined use of probes 121 and 125, which corre-
spond to the overlapping hexapeptides shown at the top of
Fig. 7, should detect only those homeobox genes that encode
the amino acid sequence Gln-Val-Lys-Ile-Trp-Phe-Gln-Asn
`!s residues 44-51 of the third a-helix of the homeodomain,
vhich is part of the putative nucleotide sequence recognition
site of homeobox proteins. Only Zen-l, zen-2, lab, and cad,
in addition to the homeobox genes described in this report
would be expected to give positive autoradiographic signals
with both 121 and 125 probes. It is uncertain whether msh,
D/l. and Abd-B genes would give positive signals with 121 and
125 probes because both Dll and Abd-B genes contain introns

UK-3 4-k UK-1
UK-b    (93E3-S)
(93El-3)

FIG. 6.  (A) In situ hybridization of genomic DNA probes for
NK-1 (first panel), NK-4 (second panel), NK-3 (third panel). NK-1
and NK-3 (fourth panel), and NK-2 (fifth panel) to Drosophila
polytene chromosomes. A-F and vertical markers represent chro-
mosomal band subdivisions in the 93 A-F region of the right aim of
the third chromosome. The DNA probes hybridize to the following
locations: NK-1, 93E3-5; NK-4, 93El-3; NK-3, 93El-3; NK-1 and
NK-3,93El-5; NK-2, lCl-5. Arrowheads indicate labeled chromo-
somal bands. (B) The approximate locations of the NK-3, NK-4, and
NK-1 genes are indicated on Bridges' revised map of chromosomal
bands (24).

of unknown sequence between codons for the 44th and 45th
homeobox amino acid residues, and it is not known whether
the msh gene contains an intron at this site. Probe 125
hybridizes to the NK-1 gene because the 3'-terminal nucle-
otide sequences of both exon 1 and intron 2 are CAG. Both
Zen-f and Zen-2 genomic DNA were detected and cloned with
probes 121 and 125 in addition to NK-1, NK-2, NK-3. and
NK-4, but lab and cad genes were not detected.

1 `:~n';,:i~C?nnG;nTTCrRC,T:TCT.~. rTTRTTS;.~GT'TT'^nF,,~~~~~~*~~,C,,~GT~  .j : :

Y" ;xT~,AI;;:,P,AcI;~. ~CnT~GTTCThGTTC_TT;T-?                :<F.

FIG. 5.  Nucleotide sequence and deduced amino acid sequence
of the homeobox region of the NK-4 gene. The homeobox is enclosed
in a box. A CAX repeat is underlined. which encodes repetitive Gin
residues. Arrowhead represents a potential splice acceptor site.

The Drosophila genome has been screened many times
with DNA fragments containing homeobox sequences as
probes; however, NK-1, NK-2, NK-3, and NK-4 homeo-
domains may not have been detected because their overall
homology to other Drosophila homeodomains is relatively
low. The use of oligodeoxynucleotide probes that correspond
to other amino acid sequences in homeobox proteins should
provide a means of detecting additional sets of homeobox
genes that have some structural features in common.

1.
2.

3.
4.

5.
6.

7.

Gehring. W. 1. (1987) Science 236, 1245-1252.
Niisslein-Volhard. C., Frohnhofer. H. G. & Lehmann. R. (1987) Scianre
238, 1675-1681.
Scott, M. P. & Cat-ml. B. C. (1987) Cell 51, 689-698.
Herr, W., Sturm, R. A., Clew R. G.. Corcoran, L. M., Baltimore, D..
Sharp. P. A.. Ingraham. H. A.. Rosenfeld. M. G.. Finney. M.. Ruvkun.
G. & Horvitz. H. R. (1988) Genes Dev. 2, 1513-1516.
Doe, C. Q. & Scott. M. P. (1988) Trends Neurosci. 11, 101-106.
Doe, C. Q., Hiromi, Y., Gehring, W. 1. & Goodman. C. S. (1988)
Science 239, 170-175.
Blochlinger. K.. Bodmer. R., Jack. J., Jan. L. Y. &Jan. Y. N. Nature
(London) 333. 629-635.


7720   Biochemistry: Kim and Nirenberg                   Proc. Nn11. Acad. Sci. USA 86 (1989)

NK-1
NK-3
NK-4
NK-2

msh
Dll
lab
Zen-l
zen-2
bed
Dfd
scr
It2

Antp
Ubx
abd-A
Abd-8
en
lnv
BSH4
BSH9
r'rd
ro
cad
H2.0
BVB
cut

     1 1    I     I     2           3   I
    1127       Z9-N ENR                Qvrrrwp   11125
    1126    ZLZZZT                     lcIEI)TpQN Y121
1       10       21       28      38  42dkXV& RTKWKKQi'
PRPARTAFT YEQLVS- TTRYLS SL SLT ET
KXRSR4AFS EA OTZLZRWA QQRYLS WIRsEb3AKSL RLT ETQVIUWFQNR RYKTKRKQI
KRKPRVLFS QAPVLPYCRPR LKKYLT -I- NLS ATQVKI~QNR RYKSKRGDI
KRKRRVLFT w QQRYLS APEREELULI RLT PTQV?XWFQNIi RYKTKPAQN

NRKPRTPFT TQQLULZZIWR EKOYLS I)LERAEpSSSL RLT ZTQVKXWFQNR RAKAKRLQE
MRKPRTIYS BL QL%X3XUQ RTQYLA LPXRULAAS L GLT QTQVKIW9'QNR RSKYKKMMK
NNSGRTNFT NXQL TXLXKWE FNRYLT RARRIPIUTL QLN ZTQVZIWPQNR RMKOKKRVK
LKRSRTAFT EVQIMLernm. SNMYLY RRwnIAQFtL SLC ZRQVZIR5-QNR RMKFKKDIQ
SKRSRTAFS SLQL IUZR.UE LNKYLA RTRRIoISpRL ALT ZRQVKIW5'QNR RMKLKKSTN
PRRTRTTFT SSQIAXIZQHJL QGRYLT APRIJQLSAKL ALG TAQVKI- RRRHKIOSD
PKRPRTAYT RBQI LKLURE YNRYLT RBRXfXzABR. VLS ZRQIKI~QNR RMKWKKDNK
TKRQRTSYT RY QTuLIxna FNRYLT RRRRIZIAEAL CLT ZRQIKI~QNR RMKWKKEHK
SKRTRQTYT RY Q-E FNRYIT RIUUUDIANU SLS IRQI-QNR RMKSKKDRT
RKRGRQTYT RYQ- FNRYLT RIUUUXIAUL CLT IRQIKIUIQNR RMKWKKENK
RRRGRQTYT RYQ- TNHYLT m CLT WQ-QNR RMKLKKEIQ
RRRGRQTYT WQlZUXKXXIi FNHYLT RRRRIZIAEAL CLT ZRQIKIWQNR RMKLKKELR
VRKKRKPYS =QT LLWWL FNAYVS KQXXm QLT ERQVKIWS-QNR RMKNKKNSQ
EKRPRTAFS SE- ENRYLT ZPRFtQQLSSZL GLN aIxI1IpQNZ RAKIKKSTG
DKRPRTAFS CYQ- ENRYLT XXRRQQLBCU GLN EJiQIKI~QNK RAKLKKSSG
QRRSRTTFT AEQLKUEIUJS RTQYPD VYTRZZUQlV ALT IURIQVWZSNR RARLRKHSG
QRRSRTTFS NDQ IMLKKIPA RTQYPD VYTRZKUQST GLT EllRvpvApSNR PARLRKQLN
QRRCRTTFS ASQID gLERlllp RTQYPD I YTREELAQRT NLT X.AIUQVWSSNR RARLRKPIIT
QRRQRTTFS TZQT- RNEYIS RSRRPPLAPTL RLT KTQIXIWYQNR PAKDKRIEK
KDKYRWYT D? QRLZUXXYC TSRYIT IRRZSZLAQTL SLS ZRQVZIUTQNR RAKERTSNK
RSWSPAVFS NLQP.KCmIQTQ QQKYIT FX'DRRKLMRL NLT DmQNR RMKWRHTRE
VRRYRTAFT RDpm KENYVS RPRRCELAAQL NLP ESTmQNFt RMKDKRQRI
SKKQRVLFS XIQKULUAT A LDPYPN VCTIKTLANKL GLATRTIRRIWIB RMRLKCQVP
00.00 . 000 .     0  00 0 .  0 000o..cm0 . 0 00

PERCENfHOMOLOClY

NK.l NK-3 NK-4 NK.2

100 54 44 49
54 100 59 66
44   59 100 59
49 60 59  100

57 54 54 54   I291
53 51 40 40   [221
51 40 40 43   119, 201
57 51 44 39   f151
49 54 40 41   1151
46 41 39 43   I261
53 48 44 30   132)
49 40 44 41   I331
44 43 44 30   1301
49 40 43 41   [34,351
40 46 43 41   [36,371
49 46 44 41   I301
46 46 44 46   1211
46 43 30 34   (161
49 46 39 39   (391
44 41 33 34   [401
40 44 30 30   I401
44 34 30 30   I251
51 51 44 46   1st 91
43 41 44 39   141)
39 46 41 39   1421
41 36 34 33   143,441
31 28 33 31   171

FIG. 7.  Comparison of the amino acid sequences (single-letter code) of NK-1, NK-2, NK-3, and NK-4 homeoboxes with known Drmophilo
homeoboxes. The positions of three a-helices identified by Otting et al. (31) in a peptide containing the Anfp homeobox are indicated by boxes
l-3 above the sequences and the corresponding amino acid residues are shown in boldface type. Oligodeoxynucleotide probes 121-127
correspond to the amino acid sequences shown at the top. Arrowhead above the NK-1 sequence represents the location of introns in NK-1.
lab. D/I, and Abd-B genes. Chromosomal clusters of homeobox genes are NK-1, NK-3, and NK-4; lab-Anfp; Ub.r-Abd-B; en and in\,; and BSH-4
and BSH-9 (gsb). Symbols at the bottom of the table represent the following for Drosuphilu homeoboxes: o . invariant amino acid residues; C.
strongly conserved amino acid residues. The percentage homology of the amino acid sequences of NK-1, NK-2. NK-3, or NK-4 homeoboxes
compared with other Drosophila homeoboxes are shown on the right. Values represent percentage of amino acid residues that are identical in
each pair of homeoboxes compared; 100% corresponds to 61 amino acid residues. Numbers in brackets are references.

8.

9.
10.


11.


12.


13.


14.


15.


16.


17.


18.


19.
20.


21.


22.


23.


24.
25.

Saint, R., Kalionis, B., Lockett, T. J. & Elizur, A. (1988) Narure
(Londonj 334, 151-154.
Tomlinson, A., Kimmel, 8. E. & Rubin, G. M. (1988) Cell 55, 771-784.
Maniatis, T., Fritsch. E. F. & Sambrook, J. (1982) Molecular Cloning:A
Laboratory Monucrl(Cold Spring Harbor Lab., Cold Spring Harbor, NY).
Maniatis, T., Hardison, R. C., Lacy, E.. Lauer. J., O'Connell, C., Quon,
D., Sim. G. K. & Efstratiadis. A. (1978) Cell 15, 687-701.
Wood, W. I.. Gitschier. J.. Laskey, L. A. & Lawn, R. M. (1985) Proc.
Nod. Acad. Sci. USA 82, 1585-1588.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acod. SC;.
USA 74, 5463-5467.
Pardue. M. L. (1986) in Drosophila: A Practical Approach, ed. Roberts,
D. B. (IRL, Oxford), pp. 111-137.
Rushlow, C., Doyle, H., Hoey. T. & Levin, M. (1987) Genes Dev. 1,
1268-1279.
Poole, S. J., Kauvar, L. M., Dress, B. & Kornberg, T. (1985) Cell 40,
37-43.
Biggin, M. D., Bickel. S., Benson, M., Pirrotta. V. &Tjian, R. (1988) Cell
53, 713-722.
Miiller, M., Affolter, M., Leupin, W., Otting, G., Wiirthrich, K. &
Gehrina. W. J. (1988) EMBO J. 7. 4299-4304.
Mldozik, M., Fjose, A. & Gehring, W. J. (1988) EMBOJ. 7, 2569-2578.
Hoey, T.. Doyle, H. J., Harding, K.. Wedeen, C. & Levin, M. (1986)
Proc. Nntl. Acad. Sci. USA 83-4809-4813.
DcLorenzi, M., Ali. N., Saari, G., Henry, C., Wilcox, M. & Bienz. M.
(1988) EMBO J. 7, 3223-3231.
Cohen, S. M., Brdnner, G., Kiittner. F., Jiirgens, G. & Jlckel, H. (1989)
Nature (London) 338, 432-434.
Sunkel, C. E.&Whittle, J. R. S. (1987) WilhelmRoux's Arch. Dev. Biol.
1%. 124-132.
Bridges, P. N. (1941) J. Hered. 32, 299-300.
Fr&rio, G., Burri. M., Bopp. D., Baumgartner, S. & Noll, M. (1986) Cell
47, 735-746.

26.

27.
28.


29.

30.
31.


32.

33.


34.

35.


36.

37.

38.


39.

40.

41.
42.

43.
44.

Berleth, T., Burri, M., Thoma, G.. Bopp. D., Richstein, S., Frigeno. G..
Nell, M. & Nusslein-Volhard, C. (1988) EMBO J. 7, 1749-1756.
Zink, B. & Pare, R. (1989) Nature (London) 337, 468-471.
Demerec, M., Kaufman, B. P., Fano, U., Sutton, E. & Sansome. E. R
(1942) Carnegie Inst. Washington Yearb. 41, 190-218.
Gehrine. W. J. (1987) in Molecular Approaches fo Developmental Biol-
ogy, eds. Firtel, R. A. &Davidson, E.`H. (Liss, New York); pp. 115-129.
Laughon. A. & Scott, M. P. (1984) Nature (London) 310, 25-31.
Otting, G.. Qian, Y., Miiller, M., Affolter, M., Gehring, W. & Wiithrich.
K. (1988) EMBO J. 7, 4305-4309.
Regulski, M., McGinnis, N., Chadwick, R. & McGinnis, W. (1987)
EMBO J. 6, 167-777.
LeMotte, P. K., Kuroiwa. A., Fessler, L. 1. & Gehring, W. J. (1989)
EMBO J. 8, 219-227.
Schneuwly, S., Kuroiwa, A., Baumgartner. P. & Gehring. W. J. (1986)
EMBO .I. 5. 733-779.
Laughon, A., Boulet. A. M., Bermingham, 1. R., Jr.. Layman. R. A. a
Scott, M. P. (1986) Mol. CeN. Biol. 6, 4676-4689.
Weinzierl. R.. Axton, J. M.. Ghysen, A. & Akam, M. (1987) Genes fIri.
1, 386-397.
Komfeld, K., Saint, R. B., Beachy, P. A., Harte, P. J., Peattie. D. A. i
Hogness, D. S. (1989) Genes Dev. 3, 243-258.
Akam, M., Dawson, I. & Tear. G. (1988) Development 104, SUPPI
123-133.
Coleman. K. G.. Poole. S. J.. Weir, M. P.. Soeller. W. C. & Kornberg.
T. (1987) Genes'Dev. 1; 19-28.
Baumgartner, S., Bopp, D., Buni, M. & Nell, M. (1987) Genes Del,. 1.
1247-i267.
Mlodzik, M. & Gehring, W. J. (1987) Cell 48, 465-478.
Barad. M.. Jack. T.. Chadwick. R. & McGinnis. W. (1988) EMBO J. 7.
2151-2161:
Macdonald, P. M., Ingham, P. & Struhl, G. (1986) CeN 47, 721-734.
Fmsh, M., Hoey, T., Rushlow. C., Doyle, H. & Levin, M. (1986) EMBcj
J. 6, 749-759.