Organization of the Hox gene cluster in the grasshopper, Schistocerca gregaria

Spreads of metaphase and interphase nuclei were made from grasshopper embryos at the 35% stage of development (16). The embryos were dissected from their eggs under locust embryo saline (150 mM NaCl/3 mM KCl/2 mM CaCl₂/1 mM MgSO₄/5 mM TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid/2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid) (Sigma)), treated with 0.02% colcemid (Sigma) for 1 hr, swollen in 1% sodium citrate for 15 min, and fixed in 3:1 ethanol/acetic acid for a minimum of 1 day at 4°C. The fixed embryos were mushed up on a siliconized coverslip in a drop of fresh 45% acetic acid, and squashed between the coverslip and a slide. The slide was submerged in liquid nitrogen until the bubbling stopped, and the coverslip flicked off. The spreads were dehydrated in 95% ethanol (3 times for 5 min each), and stored in a dry, air-tight, dark box.

Probes were made by nick translation according to standard procedures (17), to incorporate either biotin or digoxigenin. The genomic clones used are described in Dawson (18) (Scr and histone marker), Tear et al. (19) (abd-A), Kelsh et al. (20) (Abd-B), Dawes et al. (21), (SgDax), and Procunier and Smith (22) (ribosomal marker). The histone clone derives from Schistocerca americana, and the ribosomal RNA clone from D. melanogaster. All the others are from S. gregaria.

Slides were dehydrated through ethanol (5 min in each of 70%, 70%, 95%, 95%, 100% solutions) and then air-dried, followed by denaturation in 70% formamide (70°C, 2 min), and a second dehydration through an ethanol series (the first 70% ethanol being ice-cold). Denatured probe (100–150 ng), in 15 μl of hybridization mix (50% formamide/600 mM NaCl/10% dextran sulphate/2× saline sodium citrate (SSC)/1 mM EDTA/12 mM tris, pH 7.6/5× Denhardt’s solution/100 μg/ml sheared salmon sperm DNA), was sealed over the chromosome spreads with a 22 × 22 mm coverslip and rubber cement. Hybridization at 42°C was carried out overnight. The rubber cement was then removed and the coverslips washed off in 2× SSC at 42°C. The slides were washed in 50% formamide in 2× SSC (42°C, 2 times for 5 min each), followed by 2× SSC (42°C, 2 times for 5 min each), and then 4× TNFM (37°C, for 30 min and 5 min each) (4× SSC/0.05% Tween 20/5% non-fat milk powder). The sites of hybridization were detected with avidin–fluorescein isothiocyanate (Vector Laboratories) (1:500 dilution in 4× TNFM for 1 hr at 37°C), followed by 4× TNFM washes (42°C, 3 times for 5 min each), biotinylated-anti-avidin (room temperature, 20 min), 4× TNFM washes (42°C, 2 times for 5 min each), and a second layer of FITC–avidin (room temperature, 20 min). The slides were then washed in 4× TNFM (42°C, 2 times for 5 min each), followed by 4× SSC with 0.05% Tween 20 (room temperature, 2 times for 5 min each), and then 1× PBS (room temperature, 5 min), before counterstaining the chromatin with Hoechst solution (1 μg/ml in 1× PBS, at room temperature for 30 min). After a final rinse with 1× PBS (room temperature for 5 min) the spreads were mounted in Vectashield mountant (Vector Laboratories).

The signals were visualized on a Zeiss Axiophot microscope and photographed on Kodak GoldII ASA400 film. Brightness and contrast were adjusted in Adobe photoshop 3.

Testicular follicles were dissected from anesthetized adult male grasshoppers (from a population maintained in the Zoology Department, Cambridge University) under locust embryo saline. The follicles were broken up in ice-cold 1× PBS in a Dounce homogenizer with a loose-fitting plunger, and the cells separated from the rest of the follicular debris by passing through two layers of 37-μm Nitex mesh. The cells were pelleted for 5 min at 2750 × g at 4°C, and resuspended in 1 ml of ice-cold 1× PBS. The washing was repeated a total of three times. The cells were finally resuspended in L buffer (17), and warmed to 42°C for 10 min, prior to mixing with an equal volume of 2% GIBCO Ultrapure LMP agarose in water, at 42°C. Aliquots (90 μl) were pipetted into Bio-Rad plug moulds and allowed to set at 4°C for over 30 min. Each 90-μl plug contained 40–200 μg of DNA (corresponding to testicular follicle cells from 6–8 adult males). The set plugs were washed 2 times for 24 hr with 50 vol of L buffer containing 1 mg/ml proteinase K and 1% lithium dodecyl sulfate, followed by 2 times for 1 hr with 50 vol of TE buffer (pH 7.6) and 40 μg/ml phenylmethylsulfonyl fluoride, and stored in TE buffer (pH 7.6) at 4°C.

One whole plug was used for a single digest per gel lane. Plugs were washed twice for 30 min in TE buffer (pH 7.6) at 4°C, and then equilibrated with 10 vol of 1× restriction enzyme buffer at 4°C for more than 1 hr. The buffer was replaced with 3 vol of 1× restriction enzyme buffer and 80 units of restriction enzyme, and allowed to equilibrate at 4°C for more than 2 hr prior to incubation at the appropriate digestion temperature. During the incubation the enzyme and buffer were replaced, and the incubation continued for a total of approximately 20 hr. The plugs were then equilibrated with 0.5× TBE running buffer on ice for more than 2 hr.

The Hoefer HULA PFGE system was used. The running conditions were 180 V/11°C/40 hr/20–120-sec ramped pulse time, with the current being turned off while the gel reoriented.

Southern blotting was carried out as described in ref. 17, with denaturing solution (1.5 M NaCl/0.5 M NaOH) as the transfer buffer, onto Hybond N⁺ membrane (Amersham).

The probes were made by PCR. For Abd-B the probe was 558 bp of intronic sequence; for abd-A the probe was 243 bp, including part of the homeobox and a short stretch downstream; for Scr the probe was 2 kb, including the homeobox; for SgDax the probe was 660 bp of the cDNA 5′ of the homeobox; for Sgzen the probe was 508 bp, including most of the homeobox and some sequence upstream (23); and for eve the probe was 370 bp, including most of the homeobox and some upstream sequence. Primer information is available on request. Approximately 10 ng of template DNA, from a previous cold PCR reaction, was used in a reaction with [³²P]dCTP (50 μCi; 1 Ci = 37 GBq) (DuPont), and 200 μM each of dATP, dGTP, and dTTP. The amplification conditions were 95°C for 5 min, 72°C while the Taq polymerase is added, (15 cycles of 94°C for 30 sec/50°C for 30 sec/72°C for 2 min), and 72°C for 5 min. Unincorporated nucleotides were removed with a Stratagene NucTrap push column, following the manufacturer’s instructions.

Southern blot hybridization was carried out as described in ref. 17. Hybridization was for a minimum of 20 hr. Washes were at high stringency (0.1× SSPE/0.1% SDS at 65°C). Results were visualized on a PhosphorImager (Molecular Dynamics) after a 4–5 day exposure.

We were limited to using short probes because of the high frequency of repetitive sequences in the S. gregaria genome. This, together with the large size of the grasshopper genome (approximately 3 times that of human), necessitates overloading the gel to obtain a detectable signal. The resultant smiling of the bands on the pulsed field gel limits the accuracy of the size estimates to ±50 kb (estimated from several filters).

gregaria has a haploid karyotype of 11 chromosomes, distinguishable by size into three distinct groups [numbers 1–3 long, 4–8 medium, and 9–11 short (24)]. The major nucleolar organizers are localized on chromosomes 3 and 6 (25), which correspond to the major sites of hybridization of a probe against ribosomal RNA genes (Fig. 1a). Tandem arrays of histone genes are on chromosome 8, according to estimates of chromosome lengths and agreement with previous assessments (P. Bella, personal communication).

A genomic fragment from the S. gregaria Sex combs reduced gene hybridizes in situ to a single site on one of the short chromosomes that does not carry either histone or rRNA markers (Fig. 1c). Length measurements show that this is not the shortest chromosome, but either chromosome 9 or 10.

SgDax, abd-A, and Abd-B map close to Scr on the same chromosome. We have shown this by double-labeling experiments with probes for two different single-copy genes, one labeled with biotin and the other with digoxigenin, to enable simultaneous detection (Fig. 1 and data not shown). In D. melanogaster, Scr and ftz lie in the ANT-C, whereas abd-A and Abd-B are in the BX-C. Thus, genes homologous to both ANT-C (Scr, SgDax) and BX-C (abd-A, Abd-B) are closely linked in S. gregaria.

To estimate the molecular distance between these sites, the same probes were hybridized to spread chromatin from interphase nuclei. For each pair tested (Scr/abd-A, Scr/SgDax, SgDax/abd-A, SgDax/Abd-B), the sites of hybridization were separated by no more than an average of 2.6 μm. On similar spreads of human cells, this would correspond to a molecular distance of less than 2 Mb (26–28). This rough estimate is in reasonable agreement with the data from pulse field gels presented below.

To estimate the size of this Hox cluster more accurately, we digested genomic DNA with rare cutting retriction enzymes, separated the fragments by PFGE, and used Southern blot hybridization to locate the fragments carrying Hox genes.

The restriction enzyme AscI produces fragments of 100–700 kb containing grasshopper Hox genes. Despite their size, none of these AscI fragments contained more than a single one of the genes for which we have probed (Fig. 2). This means that the minimum size of the grasshopper cluster is 700 kb (summing the fragments containing Scr, SgDax, and abd-A, but excluding the fragments containing the presumed 3′ and 5′ genes).

We also wanted to find rare-cutting restriction enzymes that produced bands containing more than one Hox gene. NotI appears to be such an enzyme. NotI produces a 500–550 kb band with SgDax, abd-A, and Abd-B probes, and a 450-kb band with the Scr probe and part of a cDNA-derived from the S. gregaria zen homologue, Sgzen (23) (Fig. 2). The Abd-B probe also hybridizes to a 350-kb NotI band. The origin of the additional Abd-B band is not clear, but is probably due to polymorphism. The same probe detected single strong bands of 4.5–20 kb in digests with each of eight different six-cutter enzymes, which suggests that the gene is not duplicated. The AscI data and the NotI data together are consistent with the possibility that SgDax, abd-A, and Abd-B are on the same 500–550-kb NotI fragment, with Sgzen and Scr on an adjacent 450-kb fragment (summarized in Fig. 3). These data do not rule out the possibility that the cluster is much larger than this.

None of the above data prove that there is only a single grasshopper Hox cluster. The sequences used as probes may not be sufficiently conserved to hybridize at high stringency to paralogous Hox loci. However, two lines of evidence suggest that there is only a single Hox cluster. First, most short homeobox fragments from the Hox cluster genes hybridize to single bands on conventional Southern blots (data not shown). Second, degenerate primer PCR has provided no evidence for multiple distinct copies of single Hox classes, despite recovering three genes more than six times each (I. Millwood and K. Moyse, personal communication).

In vertebrates, both eve class homeobox genes are located adjacent to the 5′ end of the Hox clusters (corresponding to the site of the insect Abd-B gene). We used an eve cDNA obtained from S. americana (29) as a probe to see if eve is present on the same 650–700 kb AscI fragment as Abd-B; it is not (see Fig. 2). Because this AscI fragment does not contain abd-A, it probably extends well beyond the Hox cluster. However, the arrangement of the Abd-B end of the Hox cluster may be unusual (indicated by the two NotI bands), and so we cannot promote this conclusion firmly.