Studies on the Cuticlin Homologues of Brugia Species

Lewis, Emma Katherine (1997) Studies on the Cuticlin Homologues of Brugia Species. PhD thesis, University of Glasgow.

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The aim of this project was to ascertain the existence of the Brugia homologues of the C.elegans cuticlin genes, and to isolate and characterise them. Preliminary experiments in which Southern blots of cleaved genomic DNA from various parasitic nematodes was hybridised to a 32P-labelled fragent of cut-1 revealed the presence of at least one hybridising band in each of the digests, implying the presence of at least one cut- /dike gene in each of the nematode genomes. The probe hybridised to several bands in the B. pahangi digests. During the course of the project various approaches were used in the search for the Brugia cuticlin homologues. The initial approach was to screen an adult B. pahangi cDNA library with antisera raised against recombinant proteins corresponding to fragments of C. elegans CUT-1 and CUT-2. This resulted in the isolation of four clones, two of which carried the same insert, a truncated version of the other two. The full length clone is 2.2 Kb and comparison with the data base revealed it to be the B. pahangi homologue of a C. elegans dead-box ATP-binding RNA helicase gene (f01f1.7), identified during the course of the C. elegans Gerome Sequencing Project. The specific recognition of this unrelated cDNA is attributed to a combination of the following two factors: the rabbits in which the anti-cuticlin antisera were raised having a previous nematode infection resulting in the pesence of antibodies to nematode RNA helicases in the 'pre-immune' rabbit sera; and the high representation of RNA helicase cDNA species. in the adult library, due to the the role of these enzymes in translation. PCR using primers based on the sequence of the Ascaris cut-1 gene (ascut-1) was carried out on B.pahangi genomic DNA, and resulted in the isolation of a 358bp fragment of a B. pahangi cut-1-like gene, subsequently named bpcut-1, showing a 71% nucleotide homology and a 96% amino acid homolgy with the corresponding region of the C. elegans cut-1 gene, cecut-1. Primers based on the sequence of bpcut-1 were used to isolate the corresponding cDNA from B. pahangi day 7 p.i. cDNA (this time-point in the life-cycle corresponds to the L3-L4 moult), revealing that bpcut-1 has an intron not present in the cut-1-like genes of the other nematodes studied. Screening a B. malayi genomic library with bpcut-1 resulted in the isolation and subsequent sequencing of bmcut-1, a 613bp clone which is the B. malayi homologue of bpcut-1. There are three introns in the clone, and when they are removed the coding sequence corresponds to that of bpcut-1, resulting in a 97.5% homology between the two amino acid sequences. Screening a B. pahangi genomic library with bmcut-1 resulted in the isolation of 4 clones which cover the same 17kb region of the B. pahangi genome. The presence of the bpcut-1- specific intron in all four clones allows them to be identified as bpcut-1, rather than bpcut-1-like. PCR using one of the genomic clones with a 5' primer based on the linker region of EMBL3 (in which the cDNA library was made) and a 3' primer based on the bpcut-1 sequence, allowed the direction of transcription of the gene to be determined, and enabled the gene to be positioned within the genomic clone. A 948 by clone was isolated from an adult B. pahangi cDNA library by screening with bmcut-1. Sequencing and analysis revealed the clone to represent a mis-transcription of the bpcut-1 gene; the resultant cDNA is apparently the result of transcription of the non-coding strand of the bpcut-1 RNA. The pattern of bpcut-1 mRNA abundance throughout the mammalian stages of the life-cycle of the parasite strongly implies that it encodes a component of the nematode cuticle: peaks of abundance occur before the L3-L4 and the L4-adult moults, when the components of the cuticle are being maximally synthesised. The mRNA abundance profile also shows that the bpcut-1 transcript can be detected during the intermoult period, but at a very much lower abundance. There seems to be little or no bpcut-1 transcript detected in the adult mRNA, implying that the protein encoded by bpcut-1 is incorporated into the insoluble component of the cuticle at the time of cuticle synthesis, and that it is not renewed throughout the adulthood of the parasite. Localisation with an antiserum raised against recombinant ASCUT-1 showed a cuticular pattern on all life-cycle stages tested. Immunogold labelling showed that there was no labelling of the external surface of the cuticle. Interestingly, there was also strong recognition of an epitope on the surface of mature mf, implying the presence of an embryonic cut-1-like gene which was not detected by RT-PCR using bpcut-1-specific primers.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Eileen Devaney
Keywords: Veterinary science, Parasitology
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-75414
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 20:11
Last Modified: 19 Nov 2019 20:11

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