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doi: 10.1242/10.1242/dev.00320

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Functional specificity of the nematode Hox gene mab-5

Max-Planck Institut für Entwicklungsbiologie, Abt. Evolutionsbiologie, Spemannstr. 37-39, D-72076 Tübingen, Germany

View larger version (39K): [in a new window]   Fig. 1. Comparison of Hox cluster organization and sequence between C. elegans and Drosophila. (A) Drosophila contains eight Hox genes, organized into two subclusters. In C. elegans, the four core members are in one cluster, interspersed by many other unrelated genes. Two additional Hox genes, php-3 and nob-1, are located elsewhere in the genome. ceh-13, the labial ortholog of C. elegans, is not in a distal position in the cluster as it is in other organisms. (B) Hox gene organization and sequence identity (for the different parts of the proteins) of orthologous and paralogous genes at the amino acid level. Hox genes can be subdivided into the N-terminal and the C-terminal parts. Both, hexapeptide (hp) and homeodomain are localized in the C-terminal part of the protein. Within the homeodomain itself, the N-terminal arm (Na) and helices I, II and III (HI, HII, HIII) can be distinguished. The C-terminal end refers to the protein-coding part between the end of the homeodomain and the end of the protein. The numbers to the left indicate percentage of overall sequence similarity; the three following numbers refer to the sequence similarity in the N-terminal part (second number), the hexapeptide and homeodomain region (third number) and the C-terminal end (last number), respectively. (C) Amino acid sequence comparison of the Hox proteins shown in B in the homeodomain region. Black, sequence identity; gray, sequence similarity. (D) Amino acid sequence comparison of Cel-mab-5 and Ppa-mab-5 over the complete protein.

View larger version (65K): [in a new window]   Fig. 2. Morphology and cell lineage of C. elegans rays. (A) Ray morphology in an adult male. Lateral view. (B) The nine pair of rays (R1-R9) are formed by the blast cells V5, V6 and T. Anterior is towards the left and dorsal is upwards.

View larger version (15K): [in a new window]   Fig. 3. Summary of ray rescue experiments: part I. All transgenic animals were analyzed in the strain CB3531 carrying the null allele Cel-mab-5(e1290), which forms no V-rays. At least three transgenic lines were tested for each construct. Black rectangles, coding exons; stippled rectangles, introduced frame-shift mutation; X, the unique restriction site in pAG2 and all further constructs. Inserted cDNAs in pAG4 and pAG5 are indicated. n, number of animals analyzed.

View larger version (64K): [in a new window]   Fig. 4. Ray morphology in Cel-mab-5(e1290) animals rescued with various constructs. (A) CB3531, no V-rays are formed. (B) pAG4, a Cel-mab-5 cDNA rescues ray formation. (C) pAG5, a Ppa-mab-5 cDNA also rescues ray formation. (D) pAG13, an in vitro mutagenized protein containing the N-terminal arm and helix I of Cel-MAB-5 in an otherwise Cel-LIN-39 protein rescues ray formation. (E) pAG14, an in vitro mutagenized protein containing helix II of Cel-MAB-5 in an otherwise Cel-LIN-39 protein shows poor rescue of ray formation. Anterior is towards the left and dorsal is upwards.

View larger version (110K): [in a new window]   Fig. 5. Ray morphology in P. pacificus wild-type (A) and Ppa-mab-5(tu34) mutant animals (B). The spacing and position of wild-type rays (arrows) is different from the one in C. elegans. Two rays open laterally (arrowheads). R7-R9 are clustered at the tip of the animal. R1-R6 are absent in Ppa-mab-5. R7-R9 are present (arrow in B) but not all rays are visible in this plane of focus. Anterior is towards the left and dorsal is upwards.

View larger version (17K): [in a new window]   Fig. 6. Summary of ray rescue experiments: part II. All transgenic animals were analyzed in the strain CB3531 carrying the null allele Cel-mab-5(e1290), which forms no V rays. At least three transgenic lines were tested for each construct. Only the cDNAs that have been inserted into the unique restriction site of pAG3 are shown. mab-5 (blue), lin-39 (red), egl-5 (pink) and Ubx (gray), as well as the other Hox genes are shown with an individual color code, using specific darker colors for the hexapeptide and the helices of the homeodomain. The region of GFP fused to mab-5 is shown in green. See text for details.

View larger version (9K): [in a new window]   Fig. 7. Summary of vulva rescue experiments. (A) Organization of the basic Cel-lin-39 construct pKG11 (Garndien and Sommer, 2001). The construct contains more than 10 kb upstream region, all exons and introns and the 3'-UTR of Cel-lin-39. As in the case of Cel-mab-5, the endogenous gene has been silenced by a frame-shift mutation and cDNAs can be inserted into a unique SmaI (S) site. (B) The four constructs with Cel-lin-39, Cel-mab-5 and chimeric cDNAs inserted into pKG11. See text for details. All transgenic animals were analyzed in the strain MT4498 carrying the null allele Cel-lin-39(n1880), which is completely egg-laying defective. At least three transgenic lines were tested for each construct.