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. 2010 Jan;137(1):159-71.
doi: 10.1017/S0031182009990321. Epub 2009 Aug 28.

daf-7-related TGF-beta homologues from Trichostrongyloid nematodes show contrasting life-cycle expression patterns

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daf-7-related TGF-beta homologues from Trichostrongyloid nematodes show contrasting life-cycle expression patterns

H J McSorley et al. Parasitology. 2010 Jan.

Abstract

The transforming growth factor-beta (TGF-beta) gene family regulates critical processes in animal development, and plays a crucial role in regulating the mammalian immune response. We aimed to identify TGF-beta homologues from 2 laboratory model nematodes (Heligmosomoides polygyrus and Nippostrongylus brasiliensis) and 2 major parasites of ruminant livestock (Haemonchus contortus and Teladorsagia circumcincta). Parasite cDNA was used as a template for gene-specific PCR and RACE. Homologues of the TGH-2 subfamily were isolated, and found to differ in length (301, 152, 349 and 305 amino acids respectively), with variably truncated N-terminal pre-proteins. All contained conserved C-terminal active domains (>85% identical over 115 amino acids) containing 9 cysteine residues, as in C. elegans DAF-7, Brugia malayi TGH-2 and mammalian TGF-beta. Surprisingly, only the H. contortus homologue retained a conventional signal sequence, absent from shorter proteins of other species. RT-PCR assays of transcription showed that in H. contortus and N. brasiliensis expression was maximal in the infective larval stage, and very low in adult worms. In contrast, in H. polygyrus and T. circumcincta, tgh-2 transcription is higher in adults than infective larvae. The molecular evolution of this gene family in parasitic nematodes has diversified the pre-protein and life-cycle expression patterns of TGF-beta homologues while conserving the structure of the active domain.

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Figures

Figure 1
Figure 1. Primer design and amplification of tgh-2 sequences
A. Schematic of H. contortus tgh-2 genomic organisation, in comparison to the corresponding organisation of the A. caninum homologue, Ac-tgh-2, according to Freitas and Arasu (Freitas & Arasu, 2005). Based on cDNA sequence of H. contortus tgh-2 (accession number FJ391183), exons were identified on contigs 0077796, 016772 and 001624. Nucleotide lengths of exons (below or above box, bold) and introns (within box, italic) are given. Intron boundaries invariably conformed with the GT....AG consensus. Longer intronic tracts which contain gaps in existing sequence are indicated with broken lines. Note that no genomic sequence yet corresponds with exon VI of Hc-tgh-2. The active domain is encoded within Exons VII-X (indicated in yelllow). The positions of forward and reverse primers used for RT-PCR analysis (see Fig. 5 below) are indicated beneath the H. contortus diagram. B. Alignment of the active domains (AD) of TGF-β family members from C. elegans (Ce-DAF-7, AAC47389), B. malayi (Bm-TGH-2, AF104016), A. caninum (Ac-TGH-2, AAY79430 and AAX36084, identical depositions), and the newly-identified H. contortus (Hc-TGH-2, accession number FJ391183). Numbering corresponds to the Ce-DAF-2 sequence. Identical amino acids are shaded, and the amino acid sequences coded by the degenerate primers shown in solid blocks. C. Degenerate primer sequences used in this study (Y = C/T, R = A/G and N = A/C/G/T). D. Ethidium bromide gels of degenerate PCR products amplified from H. polygyrus, N. brasiliensis and T. circumcincta. Sizes of marker polynucleotides are given in bp. H. polygyrus and N. brasiliensis products were amplified using F1 and R1 degenerate primers, while the T. circumcincta product was amplified using F2 and R1 degenerate primers as shown in B.
Figure 2
Figure 2. 5’ and 3’ RACE products of TGF-β homologues
Upper panel : 5’ RACE of H. contortus, H. polygyrus, N. brasiliensis and T. circumcincta adult cDNA, amplified and visualised by ethidium bromide staining of agarose gels. Following sequencing, the indicated bands were found to contain tgh-2-like sequences. Lower panel : 3’ RACE from the same cDNA samples as above.
Figure 3
Figure 3. Full amino acid sequences and alignment of TGF-β homologues
Protein alignments of known TGF-β homologues from Homo sapiens, B. malayi, A. caninum, and novel TGF-β homologues from H. contortus, H. polygyrus, N. brasiliensis and T. circumcincta. Numbering corresponds to the human protein sequence. Predicted signal sequences are shown in red boxes, or where no signal sequence is predicted, start methionines are indicated in solid red boxes. Solid blue box indicates tetrabasic protease cleavage site, yellow boxes indicate conserved cysteines. Open turquoise boxes denote potential N-glycosylation motifs (N-X-S/T). Accession numbers for novel sequences are: Hp-tgh-2 FJ410912, Nb-tgh-2 FJ410912 and Tc-tgh-2 FJ410912.
Figure 4
Figure 4. Phylogenetic tree of TGF-β superfamily members
Neighbour Joining phylogenetic tree of TGF-β superfamily members, generated using with Poisson correction on MacVector; numbers indicate bootstrap values (percentage of calculated trees branching at each point, based on 1000 repetitions. Where no numbers are shown, bootstrap values are below 50%.
Figure 5
Figure 5. Expression levels of tgh-2 transcript in different life cycle stages
Levels of tgh-2 and actin transcription were measured by real-time PCR in different developmental stages of H. polygyrus (A), N. brasiliensis (B), H. contortus (C) and T. circumcincta (D). The genomic position of the RT-PCR primers used for H. contortus are shown in Figure 1 (RT-F and RT-R). The relative transcription levels were calculated by dividing the relative levels of tgh-2 transcription by relative levels of actin transcription. Standard errors shown are calculated from replicate determinations of tgh-2 levels divided by the mean of replicate determinations of actin levels. Relative levels of T. circumcincta tgh-2 were very similar when β-tubulin was used as the reference gene (data not shown). Data are representative of at least two independent experiments in each case.

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