Russian Journal of Developmental Biology, Vol. 32, No. 3, 2001, pp. 183–191. Translated from Ontogenez, Vol. 32, No. 3, 2001, pp. 225–233.
Original Russian Text Copyright © 2001 by Andreeva, Kuk, Korchagina, Eikem, Dondua.
GENETICS OF DEVELOPMENT
Cloning and Analysis of Structural
Organization of Hox Genes
in the Polychaete Nereis virens
T. F. Andreeva*, Ch. Kuk**, N. M. Korchagina*, M. Eikem**, and A. K. Dondua*
*Biological Institute, St. Petersburg State University, Universitetskaya nab. 7/9, St. Petersburg, 199034 Russia
**Laboratory of Development and Zoology, Zoological Museum of Cambridge University,
Cambridge CB2 3EJ, Great Britain
Received December 21, 1999; in final form, August 20, 2000
Abstract—We have undertaken an active search for homeobox-containing sequences of Antp class (Hox genes)
in the genome DNA of polychaete Nereis virens. This search was based on the high evolutionary conservation
of these sequences, which made possible their amplification in the polymerase chain reaction with degenerate
primers. As a result, eleven fragments of various Hox genes, including AbdB-like Nvi-post1, were cloned. Using
pulsed-field electrophoresis, we have demonstrated that Hox genes corresponding to the isolated fragments are
clustered in the genome of N. virens.
Key words: polychaete, homeobox-containing genes, homeobox structure, cluster organization, evolutionary
conservatism
Homeobox-containing (Hox) genes were originally
studied in detail in Drosophila by Lewis (Lewis, 1978).
Later, these genes were found in all animal species
studied in this respect. In evolutionarily advanced animals, their functions are diverse and varied. They control numerous morphogenetic developmental programs
at various levels. Thus, Hox genes can serve as regulators of several gene clusters, including those controlling proliferation and differentiation (Martinez et al.,
1997; Dondua, 1997; Weatherbee et al., 1999). The
main function of Hox genes, which probably appeared
before the divergence of protostomes and deuterostomes (Akam, 1995), is the specification of body parts
along the anterior-posterior axis of the body. This function is an inherent characteristic of all animals with
bilateral symmetry, which determines the high evolutionary conservatism of this gene cluster. At the same
time, the problem of the ancestral function of Hox
genes has not been examined.
Hox genes are organized in clusters in the genomes
of various animals; this organization may reflect their
origin. Three groups of genes (anterior, central, and
posterior) can be distinguished within Hox clusters;
they vary in their origin, genetic relations, and functions (Finnerty and Martindale, 1998; De Rosa et al.,
1999). In evolutionary terms, a Hox cluster is not a stable structure. Most characteristic modifications include
the loss or duplication of individual members of the
cluster, duplications and breaks within the cluster, loss
of individual clusters, or partial inversion within the
cluster (Holland, 1997; De Rosa et al., 1999).
It is very likely that evolutionary transformations of
Hox clusters, including modifications in the regulatory
regions of Hox genes, underlie evolutionary morphologic changes in animals. A comparative analysis of the
organization of Hox genes and Hox clusters in different
types of animals, including those with a primitive morphology, is necessary for a proper understanding of
such changes. This explains our interest in the organization of this gene group in the polychaete worm
N. virens. Cloning, structural analysis of evolutionaryconservative fragments of Hox genes, and proof of the
existence of this gene cluster are the first steps in understanding their morphogenetic and evolutionary significance.
MATERIALS AND METHOD
N. virens DNA was isolated from the sperm
obtained from N. virens males during spawning in late
June or early July. The work was conducted at the
Marine Biological Station of St. Petersburg State University (Island Srednii, Chupa inlet of Kandalaksha
Bay in the White Sea). Undiluted sperm was stored in
50% glycerol with 0.125 M EDTA (pH 7.5) at 20°C.
1062-3604/01/3203-0183$25.00 © 2001 MAIK “Nauka /Interperiodica”
184
ANDREEVA et al.
DNA preparation. N. virens sperms were rinsed of
the glycerol and used for DNA isolation by the standard
technique of deproteinization.
Cloning of homeobox-containing fragments of
Hox genes. We used kits of direct and reverse prim-
ers for polymerase chain reaction (PCR) complementary to the most conservative fragments of the
homeobox. Primers AN1 and AN2 were kindly provided by Dr. P. Martinez (California Technology
Institute, USA).
Direct primers:
E
L
E
K
E
GAR
CTI
GAR
AAR
GAR
TT
Fbam
CTI
GAR
CTI
GAR
AAR
GA
AN1
YTI
GAR
YTI
GAR
AAR
GAR
T.
W
I
K
RAA
CCA
1798N
F
Reverse primers:
R
K
N
Q
F
1797I
ICG
RTT
TTG
RAA
CCA
Rxba
YTT
CAT
ICG
ICG
RTT
Rend CG
YTT
RTT
TTG
RAA
CCA
AN2
ATT
CAT
ICK
ICK
RTT
Thus, we had an opportunity to perform PCR with
the genome DNA of N. virens using 12 various combinations of the primers.
Preparation of Hox gene fragments by the method of
inverted PCR (IPCR). IPCR allows identification of
DNA sequences adjacent to a known sequence. N.
virens DNA was digested with one of the following
restriction endonucleases: Eco RI, Bam HI, Hind III,
Alu I, or any other frequently cleaving enzyme. DNA
fragments obtained were ligated, and the resulting circular DNA were used as templates for PCR. Pairs of
nested primers for each fragment of Hox gene cloned
with the use of degenerate primers were constructed
in such a way that the ends of the amplified
IPCR fragment were in the middle of an earlier
known sequence.
Analysis of amplified fragments was performed in a
2% agarose gel. Amplified fragments of the required
length were extracted from the gel and cloned in the
BlueScript SK+ plasmid in Eco RV restriction site.
Recombinant clones were selected on the medium with
YTG
YTG
IAT
YT.
X-gal and IPTG. For each combination of primers, 20
to 60 white or light-blue clones were selected and
tested for the presence of the insert of the required
length by the method of “confirming” PCR. In this
case, PCR was run with T3 and T7 primers recognizing
vector sequences. About 500 different clones were analyzed in this way and 103 cloned fragments were
sequenced. Sequencing was performed using PCR with
four different fluorescent terminating dideoxynucleotide triphosphates (ddNTP) with the subsequent analysis of gels in an automatic sequencer. Comparison of
the primary sequences obtained with the GenBank
database was conducted using WU-BLASTX software.
Preparation of high-molecular-weight DNA of
N. virens. Nereis virens sperms were rinsed of glycerol
with 0.125 M EDTA solution (pH 7.5) and thereafter
mixed at 45°C with an equal volume of 1.5% agarose
(Sigma, USA) made in 0.125 M EDTA (pH 7.5). The
final concentration of DNA was 0.2 µg/µl. DNA concentration was determined according to Spirin (1958).
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 32
No. 3
2001
CLONING AND ANALYSIS OF STRUCTURAL ORGANIZATION OF HOX GENES
The suspension was poured into a standard form and
left to solidify. The prepared blocks were then transferred into a lysing solution containing proteinase K
(1 mg/ml) and incubated at 52°C for 50 h (Smith et al.,
1990). Thereafter, the blocks were incubated three
times (1 h each time) in TE buffer (10 mM Tris-HCl,
pH 7.4, 0.1 mM EDTA) containing 1 mM PMSF (phenylmethylsulfonylfluoride) at 4°C. The prepared blocks
were kept in 0.5 M EDTA (pH 9.5) at 4°C.
Treatment of DNA in agarose blocks by restriction
endonucleases was conducted according to Smith et al.
(1990). We used restriction endonucleases Not 1, Asc 1,
Sfi 1 (New England Biolabs Inc., USA).
Pulse-field electrophoresis (PFE). PFE was conducted in an instrument similar to a Pulsafor manufactured by Pharmacia-LKB (Sweden). Electrophoresis
was run for 52–54 h at 14–15°C under the following
conditions: TBE buffer (0.1 M Tris-HCl basic, 0.1 M
boric acid, 0.2 mM EDTA); 1% agarose gel 12 × 12 ×
0.4 cm; voltage 100 V; pulse time was changed over
electrophoresis: 55 s, 10 h; 110 s, 24 h; 220 s, 20 h.
Chromosomes of S. cerevisiae strain 15VP-4 were used
as a molecular mass marker (Timofeeva and Rautian,
1997).
In order to identify practically all Hox genes (about
ten of them are in the genome of invertebrates), it is sufficient to sequence about hundred various clones (Dick
and Buss, 1994). It has been demonstrated (Dick and
Buss, 1994; Irvine et al., 1997) that success of the procedure depends both on the nature of the template
(genome DNA, cDNA) and the nucleotide sequence
of primers, since due to code degeneration, the same
protein sequences can be coded by various sets of triplets.
We used 12 various primer combinations. Electrophoretic analysis of amplificates in 2% agarose gel has
demonstrated the presence of an amplified fragment of
the required length in 11 primer variants (130–140 bp,
depending on the primer), although the intensity of
amplification varied significantly.
Radioactive probes were prepared using PCR with
T3 and T7 primers (MBI Fermentas, Lithuania). dATP
with 32P (St. Petersburg) in α-position was used for
labeling.
Southern hybridization was performed according to
Maniatis et al. (1982). After autoradiography, the probe
was removed from the membrane as recommended by
the manufacturer (Amersham, Great Britain).
RESULTS AND DISCUSSION
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Amplification
rate
Primers
Southern transfer of DNA to a nylon membrane
(Hybond-N+) was conducted by the method recommended by the manufacturer (Amersham, Great Britain).
Hox genes belong to the gene group of Antp class. It
is known that the first and third/fourth α-helices in
homeodomains of class Antp proteins have the most
highly conserved regions, ELEKEF and WFQNKR,
respectively (Burglin, 1994). These conservative
sequences were used for the construction of primers for
PCR, which allow the production of amplified fragments containing in their central part homeobox-containing sequences of matrix DNA, N. virens genome
DNA in our case. Amplified fragments should ideally
contain sequences of all Antp class genes present in the
genome. By cloning amplification products and subsequent analysis of recombinant plasmids (determination
of the length and primary sequence of the cloned fragment) and comparison with the database, it is possible
to assign the cloned fragment to a specific homeoboxcontaining gene.
185
AN1
AN2
+++++
AN1
1797I
+++
AN1
Rxba
+++
AN1
Rend
+++++
1798C
AN2
+++++
1798C
1797I
++
1798C
Rxba
++
1798C
Rend
++++
Fbam
AN2
++++
Fbam
1797I
–
Fbam
Rxba
+
Fbam
Rend
++
It should be pointed out that only some primer combinations result in the intense amplification of a 130–
140 bp fragment.
Fragments of the required length were cloned in the
BlueScript SK+ plasmid. Data on the number of
selected recombinant clones and of recombinant clones
carrying a DNA fragment of the target length are given
in Table 1. As follows from the table, the yield of DNA
fragments of the expected length capable of producing
recombinant clones is different for different combinations of degenerate primers.
Vol. 32
No. 3
2001
186
ANDREEVA et al.
Characteristics of recombinant clones produced with the use
of degenerate primers
Number of clones
Primers
recom- with fragment of
binant the required length
selected for
sequencing
AN1
AN-2
40
29
29
AN1
1797I
40
0
2
AN1
Rxba
51
15
13
AN1
Rend
40
15
14
1798C AN2
63
35
17
1798C 1797I
43
1
1
1798C Rxba
34
2
4
1798C Rend
26
4
5
Fram
AN2
31
0
0
Fram
Rxba
40
8
3
Fram
Rend
59
39
1
Total:
467
148
103
Analysis of 103 primary sequences has demonstrated that 69 of them have homeobox-containing
fragments belonging to 14 different genes. Four genes
do not belong to standard clustered Hox genes and are
not discussed in this paper. Thus, we found fragments
of 10 Hox genes in the N. virens genome: (a) labial-like
(Nvi-lab), proboscipedia-like (Nvi-pc), Hox3-like (NviHox3); (b) four homeobox-containing fragments
homologous to homeobox of Drosophila gene Antennapedia, which differed in nucleotide sequences, but
corresponded to one amino acid sequence: 20 Antp-like
(Nvi-20Antp), 49 Antp-like (Nvi-49Antp), 50 Antp-like
(Nvi-50Antp) and 60 Antp-like (Nvi-60Antp); (c) fragments of genes lox2-like (Nvi-lox2), lox4-like (Nvilox4), which are most similar to genes lox2 and lox4 of
the leech; (d) a fragment of the homeotic gene
AbdB-like (Nvi-post1). As shown for Drosophila,
grasshopper, crayfish, frog, mouse, some other animals, and man, the latter gene is responsible for specifying the posterior parts of animals.
The first groups of genes were also found in other
polychaetes (Dick and Buss, 1994; Irvine et al., 1997).
Gene AbdB-like, a representative of the group of “posterior” homeotic genes, was first described by us for
the annelid worms. We found fragments of all genes
many times: Nvi-lab was found eight times; Nvi-pb,
two times; NviHox3, six times; Nvi-20Antp,
17 times; Nvi-49Antp, five times; Nvi-50Antp, seven
times, Nvi-60Antp, two times; Nvi-lox2, six times;
Nvi-lox4, four times; Nvi-post1, five times. This suggests that we have found all possible genes of
Antp class present with a high probability in the
genome of N. virens.
Below we present nucleotide and amino acid
sequences of fragments of the N. virens Hox genes
cloned by us, as well as comparative analysis of homeodomain fragments in the N. virens genome and
genomes of other animals. (Ame—Apis mellifera; Bfl—
Branchiostoma floridae; Cel—Caenorhabditis elegans; Chv—Chaetopterus variopedatus; Cts—Ct. serratus; Dme—Drosophila melanogaster; Hme—Hirudo
medicinalis; Hro—Helobdella robusta; Htr—Helobdella triserialis; Lan—Lingula anatina; Mmu—Mus
musculus; Nvi—Nereis virens; Pca—Priapulus caudatus; Pvu—Patella vulgata; Sas—Salmo salar).
Nvi-lab:
1
10
20
30
40
TTC CAT TTT AAT AAA TAC CTC ACG CGG GCG CGG AGG ATA GAA
F
H
F
N
K
Y
L
T
R
A
R
R
I
E
50
60
70
80
ATC GCA GCT GCC CTG GGA CTC AAT GAA ACA CAA GTG AAA ATC
I
A
A
A
L
G
L
N
E
T
Q
V
K
I
Nvi
Cts
Chv
Hro
Mnu
Dme
lab
lab
Hb3
Lox7
Hoxa1
lab
FNKYL TRARR I E I AAALGLNE TQV
++++++++++++++++++++++++
++++++++++++++++++++++++
+ + + + + + + + + + + + + + ST+ + + + + + + +
+ + + + + + + + + +V+ + + + S +Q+ + + + + +
+ + R + + + + + + + + + + +NT +Q+ + + + + + .
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 32
No. 3
2001
CLONING AND ANALYSIS OF STRUCTURAL ORGANIZATION OF HOX GENES
Nvi-pb:
1
10
20
30
40
TTC CAC TTC AAC AAA TAC CTA TGC CGG CCA AGA AGA ATC GAC
F
H
F
N
K
Y
L
C
R
P
R
R
I
E
50
60
70
80
ATC GCT GCT TCA CTG GAC CTG ACC GAA AGA CAG GTC AAA GTT
I
A
A
S
L
D
L
T
E
R
Q
V
K
V
Nvi
Cts
Pca
Bfl
Dme
Mmu
pb
pb
pb
Hox2
pb
Hoxa2
FH F NKYL CR P RR I E I AAS LDLT E RQVK
+ + + + + + + + + + + ++ + + + + T + + + + + K + + +
+ + + + + + + + + + + ++ + + + + + + + + + + + + + +
+ + Y + ++V+K+ + +K+ + + SY+ + +N+ + + + +
+ + + + + + + + + + + ++ + + + + + + + + + + + + + +
+ + + + ++ + + + + + +V+ + + + L + + ++ + + + + + .
Nvi-Hox3:
1
10
20
30
40
TTC CAC TTC AAT CGC TAC TTG TGT CGC CCT AGG CGG ATT GAG
F
H
F
N
R
Y
L
C
R
P
R
P
I
E
50
60
70
80
ATG GCT GCC CTG TTG AGT TTG TCA GAG AGG CAG ATC
M
A
A
L
L
S
L
S
E
R
Q
I
Nvi
Cts
Chv
Htr
Mnu
Hox3
Hox3
Hb5
Lox-20
Hoxd3
FH F NRYL CR P RR I EMAAL L S LS E RQ I
+ + + + + + + + + + + ++ + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +N+ T + + + + +
+ + + + ++ + S +R + ++ + + I + +HV+ S + + + +
+ + + + ++ + + + + + +V+ + +N+ +N+T + + + + .
Nvi-49Antp,
Nvi-20Antp,
1
TTC
TTT
TTT
TTT
F
CAC
CAC
CAC
CAC
H
Nvi-20
Nvi-49
Nvi-50
Nvi-60
ATA
ATA
ATA
ATA
I
Nvi-20
Nvi-49
Nvi-50
Nvi-60
CAG
CAG
CAG
CAC
Q/H
Nvi-20
Nvi-49
Nvi-50
Nvi-60
Nvi
Ame
Zebrafish
Mmu
Homo sapiens
Nvi-50Antp,
TAC
TTC
TTC
TTC
Y/F
10
AAC
AAC
AAC
AAC
N
CGA
AGA
AGA
CGA
R
40
GAG
GAG
GAA
GAG
E
ATC
ATT
ATC
ATT
I
GCT
GCG
GCG
GCC
A
50
CAT
CAT
CAT
CAC
H
ATC
ATC
ATC
ATC
I
80
AAG
AAA
AAA
AAA
K
ATC
ATC
ATC
ATT
I
Antp
h90
zf-22
Hoxb-6
Hoxa-7
Nvi-60Antp:
TAC
TAC
TAC
TAC
Y
20
CTG
CTA
CTC
CTG
L
ACC
ACC
ACA
ACC
T
GCG
GCG
GCG
TCC
A/S
CTT
CTG
CTG
CTG
L
60
GGG TTG
AAT TTA
TGT CTG
TGT CTG
G/N/C L
AGA
AGG
CGG
AGG
R
30
AGA AGG
AGG AGG
AGA AGA
AGG AGG
R
R
CGG
CGA
CGA
CGA
R
ACA
ACC
ACT
ACA
T
70
CGA
AGA
CGC
CGC
R
GAA
GAG
GAG
GAA
E
FHYNRYL TRRRR I E I AHALGLTERQ I K I
+ + + + + + + + + ++ + + ++ + + + + + + + + + + + + +
+ + + + + + + + + ++ + + ++ + + + + + + + + + + + + +
+ + + + + + + + + ++ + + ++ + + + + + + + + + + + + +
+ + + + + + + + + ++ + + ++ + + + + + + + + + + + + + .
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 32
No. 3
2001
187
188
ANDREEVA et al.
Nvi-lox2:
1
10
20
30
40
TTC AAA TTC AAC CGA TAT TTA ACC CGA AAG CGT CGG ATC GAA
F
K
F
N
R
Y
L
T
R
K
R
R
I
E
50
60
70
80
CTG TCT CAT ATG CTC TGT CTT ACT GAA CGT CAG ATA AAA ATT
L
S
H
M
L
C
L
T
E
R
Q
I
K
I
Lox2
Lox2
Hb9
Lox2
Lox2
Lox2
h50
s12-a
Nvi
Cts
Chv
Htr
Pvu
Lan
Ame
Sas
FK F NRYL T RKRR I E L SHMLCLT E RQ I K
+ + + + + + + + + R + ++ + + + + + + + + + + + + + +
+ + + + + + + + + R + ++ + + + + L + + + + + + + + +
+ + + + ++ + + +R + ++ + + + + T +Y++ + + + + +
+ + + + ++ + + +RV++ + + + + + + + ++ + + + + +
+ + + + + + + + + R + ++ + + + + + + + + + + + + + +
H+ + ++++ + +R+++ I A++A+++++ + + + +
H+ + ++++ + +R+V+ I A+++V++++ + + + + .
Nvi-lox4:
1
10
20
30
CGG TTC AAC CAC TAC CTA ACT CGG AAA CGA CGG ATG GAG
R
F
N
H
Y
L
T
R
K
R
R
M
E
40
ATC
I
50
60
70
GCT CAT GTG CTC TGT CTA ACC GAA CAC CAA ATC
A
H
V
L
C
L
T
E
H
Q
I
F R F NHYL T RKRRME I AHVL C LT E HQ I K
Nvi
Lox4
+ + + + ++ + + + + + + I + + + +A+ + ++ + R + + +
Cts
26632
Chv
Hb6
Q + + ++ + + + + + ++ + + + +A+ + ++ + R + + +
Hme
Lox4
+Q + + R+ + + + + + + I + + + + C + + + + + R + + +
Q + + +++ + + + + + I + + + + T + + ++ + R + + +
Pvu
Lox4
Lan
Lox4
+Q + + + + + + + + + + I + V + + A + + + + + R + + + .
Nvi-post1:
1
10
20
30
40
TAC GTG AAC AAC ACC TAT ATC ACC AAA CCA AAA CGA TGG GAA
Y
V
N
N
T
Y
I
T
K
P
K
R
W
E
50
60
70
CTG TCG CAG CGG CTC AAC CTC AGC GAA CGA CAG GTC
L
S
Q
R
L
N
L
S
E
R
Q
V
Nvi
Dme
Pca
Lan
Cel
post1
AbdB
AbdB
post1
Y75B8A1
YV N N TY I T K P KRWE L S QR L N L S E R Q V K
F L F + A+ V S + Q + + + + + A R N + Q + T + + + + +
F L F + A+ V S + Q + + + + + A R T + + + T + + + + +
+ + S + + + + S + + + ++ + + + + + +Q+ + + + + + +
F L Y + + + V S + Q + + + + + AKY + H + T + + + + + .
Thus, we have established that there are at least ten various homeobox-containing Hox genes in the genome of
polychaete N. virens. The small length of cloned
sequences did not allow us to identify Antp-like genes.
Moreover, some marker sequences for many Hox genes
are located in other parts of the homeobox or even beyond
it. Using IPCR, we have determined the primary sequence
of the full-size homeobox for eight Hox genes. Their
amino acid sequences are listed below. Dots at the left and
at the right mean that the flanking sequence is known.
Nvi-lab:
...PNMGRTNFTNKQLTELEKEFHFNKYLTRARRIEIAAALGLNETQVKIWFQNRR
MKQKKRM...
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 32
No. 3
2001
CLONING AND ANALYSIS OF STRUCTURAL ORGANIZATION OF HOX GENES
189
Nvi-pb:
...PRRLRTAYTNTQLLELEKEFHFNKYLCRPRRIEIAASLDLTERQIKI...
Nvi-Hox3:
...SKRARTAYNSAQLVELEKEFHFNRYLCRPRRIEMAALLSLSERQIKIWFQNRR
MKYKKDQ...
Nvi-Dfd(Nvi-50Antp):
...SKRTRTAYTRHQVLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRM
KWKKEN...
Nvi-Scr(Nvi-49Antp):
...SKRTRTSYTRHQTLELEKEFHFNRYLTRRRRIEIAHALNLTERQIKIWFQNR
RMKWKKEH...
Nvi-lox4:
...EERGRQTYSRYQTLELEKEFRFNHYLTRKRRMEIAHVLCLTEHQIKIWFQNR...
(intron)
Nvi-lox5(Nvi-20Antp):
...QKRTRQTYTRYQTLELEKEFHYNRYLTRRRRIEIAHALGLTERQIKIWFQNRRM
KWKKEN...
Nvi-post1:
...MRKKRKPYSKYQIAELEKEYVNNTYITKPKRWELSQRLNLSERQVKIWFQNR
RMKEKKVT...
The fragments obtained were used to prepare radioactive 32P-probes. Using Southern blot-hybridization
(Maniatis et al., 1984), we have demonstrated that they
hybridize with individual fragments of various
Hox genes, confirming that our probes are specific
(Korchagina, 1998).
One of the widely used methods of analysis of linkage of Hox genes is based on electrophoretic separation
of DNA in the pulse field (PFE). This method was used,
in particular, for the analysis of cluster organization of
Hox genes of Xenopus laevis (Dekker et al., 1993), sea
urchin (Martinez et al., 1997), and grasshopper (Ferrier
and Akam, 1996). Varying the combination of restriction endonucleases, it is possible to demonstrate not
only the localization of certain Hox genes in one fragment, but also, in some cases, their order within the
cluster.
A typical PFE pattern is shown in Fig. 1. Figure 2
shows a typical result of Southern blot-hybridization
after PFE with probes Nvi-Hox3 (2a) Nvi-Dfd (2b), NviScr (2c), and the result of the hybridization of the latter
membrane with Nvi-lab probe. Results of hybridization
and rehybridization with Nvi-lox4, Nvi-lox5, Nvi-post1
are not shown. As follows from Fig. 2, different restriction endonucleases identify different DNA fragments
on autoradiographs (for example, on Fig. 2a, this fragment has a size of ~780 kb for Sfi I, ~2600 kb for Not I,
and ~2400 kb for Asc I), but fragments for a given
restriction endonuclease are of the same size for all
genes shown in Fig. 2. We conclude that all the genes
analyzed are located in the same DNA fragment, i.e.,
they form a cluster. As follows from the results of
hybridization with Nvi-lox4, Nv-lox5, Nvi-post1, they
are located in 2400-kb fragment after digestion with
Asc I. There are no data on Sfi I. Thus, we can conclude
that all the genes analyzed by us are located in the same
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
DNA fragment 2400 kb in size. It should be noted that
in all animals studied thus far, the gene paralogous to
lab is located at the 3'-end of the cluster, while the
kb
1
2
3
4
2500
1700
1200
1000
820
760
620
530
430
330
250
200
Fig. 1. DNA of N. virens fractionated by pulse-field electrophoresis. Tracks: (1–3) native N. virens DNA subjected to
restriction by endonucleases Sfi I (1); Not I (2); Asc I (3);
(4) marker, yeast chromosomes.
Vol. 32
No. 3
2001
190
ANDREEVA et al.
1
2
3
1
2500 kb
2
3
2500 kb
1
2
2500 kb
3
1
2
3
2500 kb
760 kb
760 kb
760 kb
760 kb
(‡)
(b)
(c)
(d)
Fig. 2. Blot hybridization with the 32P probe: (a) Nvi-Hox3; (b) Nvi-Dfd; (c) Nvi-Scr; (d) rehybridization of membrane c with the
probe Nvi-lab. Tracks: N. virens DNA digested with Sfi I (1); (2) Not I (2); (3) Asc I.
genes paralogous to AbdB (Nvi-post1 in the polychaete
N. virens) are located at the 5'-end of the cluster
(De Rosa et al., 1999). This allows us to conclude that
Hox genes studied by us are clustered within a fragment
not exceeding 2400 kb in length.
ACKNOWLEDGMENTS
This study was supported by the Russian Foundation for Basic Research, project no. 97-04-48 924.
We are grateful to Dr. P. Martinez (California Technology Institute, USA), M.S. Rautian, A.S. Timofeeva,
A.V. Pinevich, and Zh.E. Fedorova (Biological Institute, St. Petersburg State University), and I.M. Bessonov for their help.
REFERENCES
Akam, Hox Genes and Evolution of Diverse Body Planes,
Phil. Trans. R. Soc. L., 1995, vol. 349, pp. 313–319.
Burglin, T.R., A Comprehensive Classification of Homeobox
Genes, Duboule, D., Ed., New York: Univ. Of Cambridge
Press, 1994, pp. 25–71.
Dekker, E.J., Pannese, M., and Houtzager, E., Colinearity in
the Xenopus Laevis Hox-2 Complex, Mech. Devel., 1993,
vol. 40, pp. 3–12.
De Rosa, R., Jennifer, K.G., Andreeva, T.F., et al., Hox Genes
in Brachipods and Priapulids and Protostome Evolution,
Nature, 1999, vol. 399, pp. 772–776.
Dick, M.H. and Buss, L.W., A PCR-Based Survey of
Homeobox Genes in Ctenodrilus serratus (Annelida: Polychaeta), Mol. Phylogenet. Evol., 1994, vol. 3, pp. 146–158.
Dondua, A.K., The Role of Homeobox-Containing Genes
Clusters in Morphogenesis of Animals, Ontogenez, 1997,
vol. 28, no. 1, pp. 3–17.
Ferrier, D. and Akam, M., Organisation of Hox Genes Cluster
in the Grasshopper, Schistocerca gregaria, Proc. Natl. Acad.
Sci. USA., 1996, vol. 93, pp. 13 024–13 029.
Finnerty, J.R., and Martindale, M.Q., The Evolution of the
Hox Cluster: Insights from Outgroups, Current Opinion
Genet. Devel., 1998, vol. 8, pp. 681–687.
Holland, P.W., Vertebrate Evolution: Something Fishy about
Hox-Genes, Current Biol., 1997, vol. 7, pp. 570–572.
Irvine, S.Q. and Martindale, M.Q., A Survey of Homeobox
Genes in Chaetopterus variopedatus and Analysis of Polychaete Homeodomains, Mol. Phylogenet. Evol., 1997, vol. 7,
pp. 331–345.
Korchagina, N.M., Analysis of Specificity of the Probes for
Hox Genes of Polychaete N. virens, Trudy Sankt-Peterburg
Univ. (Proc. St. Petersburg State University), Ipatov, V.S.,
Ed., St. Petersburg: St. Petersburg Univ. Press, 1998,
pp. 118–119.
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 32
No. 3
2001
CLONING AND ANALYSIS OF STRUCTURAL ORGANIZATION OF HOX GENES
Lewis, E.B., A Gene Complex Controlling Segmentation in
Drosophila, Nature, 1978, vol. 276, pp. 565–570.
Maniatis, T., Fritch, E., and Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,
New York: Cold Spring Harbor, 1982.
Martinez, P., Lee, J.C., and Davidson, E.H., Complete
Sequence of SpHox8 and Its linkage in the Single Hox Gene
Cluster of Strongylocentrotus purpuratus, J. Mol. Evol.,
1997, vol. 44, pp. 371–377.
Smith, K., Klko, S., and Cantor, Ch., Pulse Electrophoresis
and Methods of Work with Large DNA Molecules, Genome
Analysis. Methods, Davis, K., Ed., translated under the title
Analiz genoma. Metody, Moscow: Mir, 1990, pp. 58–93.
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
191
Spirin, A.E., Spectrophotometric Determination of the Total
Amount of Nucleic Acids, Biokhimiya, 1958, vol. 23, no. 5,
pp. 656–662.
Timofeeva, A.S. and Rautian, M.S., Determination of the
Size of Genome of Intranuclear Symbiotic Bacterium
Holospora undulata by Pulse Electrophoresis, Tsitologiya,
1997, vol. 39, no. 7, pp. 634–639.
Weatherbee, S.D., Nijhout, H.F., Grunert, L.W., et al., Ultrabithorax Function in Butterfly Wings and the Evolution of
Insect Wing Patterns, Current Biol., 1999, vol. 9, pp. 109–
115.
Vol. 32
No. 3
2001