Serine proteinases of the human body louse (Pediculus humanus): sequence characterization and expression patterns

Abstract

After the previous characterization of one trypsin gene (Try1) of the human body louse Pediculus humanus, genes encoding a second trypsin (Try2) and a chymotrypsin (Chy1) have been cloned using degenerate serine proteinase primers and 5'- and 3'-RACE, and sequenced. The deduced 259 and 267 amino acid sequences of Try2 and Chy1 show an identity of 33%-40% to trypsinogens and chymotrypsinogens of other insects. Considering previously published partial sequences, P. humanus possesses at least one Try1 gene, five variants/isoforms of Try2 and six variants/isoforms of Chy1. The genomic DNA of Try2 contains three introns and Chy1 contains five introns. Using whole mount in situ hybridization, gene expression of Try1, Try2 and Chy1 has been localized not only in the distensible anterior region of the midgut of lice but sometimes also in the area following the distensible region. The Try2 gene was always expressed at much lower levels than Try1 or Chy1. This lower expression, the constitutive expression of Try1 and Chy1 at 1, 2, 6, 12 and 24 h after feeding of adults and the regional differences have been verified in quantitative real-time PCR.

Sequence characterization and expression patterns of defensin and lysozyme encoding genes from the gut of the reduviid bug Triatoma brasiliensis

“…Based on the methodology of Tautz and Pfeifle (1989) and modifications of Wilkinson (1992) and Waniek et al (2005), defensin-or lysozyme-specific mRNA was detected in different regions of the gut by whole mount in situ hybridization. For the synthesis of RNA probes, def1 and lys1 cDNAs were cloned into the pGEM-T Easy vector (Promega, MD, USA).…”

“…For each reaction, 0.2 mg of total RNA was used. Details of the procedure have been published previously (Waniek et al, 2005). The standard curves for the absolute quantification of the specific mRNA were generated with different quantities (10,000, 1000, 100, 10, 1 and 0.1 pg) of the respective cDNA encoding defensin or lysozyme (Meijerink et al, 2001).…”

“…For the synthesis of RNA probes, def1 and lys1 cDNAs were cloned into the pGEM-T Easy vector (Promega, MD, USA). These recombinant plasmids were the templates for PCR with M13-forward and -reverse oligonucleotides (Waniek et al, 2005). The resulting PCR products containing the amplified SP6-and T7-RNA polymerase promoter sequences were used as templates for the synthesis of sense and antisense digoxygeninlabelled RNA probes with the T7-and SP6-RNApolymerases.…”

“…For the detection of labelled probes, antibodies combined with alkaline phosphatase were used and stained for 3 h with Fast Red TR/Naphthol (Sigma, Deisenhofen, Germany) in alkaline phosphatase buffer (Waniek et al, 2005).…”

Sequence characterization of an unusual lysozyme gene expressed in the intestinal tract of the reduviid bug Triatoma infestans (Insecta)

“…Gene-specific primers for the 5′-RACE reaction were 5′-AGTAATAAACG CCATCAAA-3′ and 5′-AACGCCATCAAAGTCTGA-3′, and the specific primer for 3′-end amplification was 5′-ATGAAAGCTATTTTATTACTATGCC-3′. The cDNA products were amplified by conventional PCR using GoTaq Polymerase (Promega; Waniek et al 2005). Cloning of the PCR products was carried out with the pGEM T-vector system (Promega), and sequencing was conducted by MWG-Biotech (Ebersberg, Germany).…”