Expressed sequence tags analysis of Blattella germanica
Article information
Abstract
Four hundred and sixty five randomly selected clones from a cDNA library of Blattella germanica were partially sequenced and searched using BLAST as a means of analyzing the transcribed sequences of its genome. A total of 363 expressed sequence tags (ESTs) were generated from 465 clones after editing and trimming the vector and ambiguous sequences. About 42% (154/363) of these clones showed significant homology with other data base registered genes. These new B. germanica genes constituted a broad range of transcripts distributed among ribosomal proteins, energy metabolism, allergens, proteases, protease inhibitors, enzymes, translation, cell signaling pathways, and proteins of unknown function. Eighty clones were not well-matched by database searches, and these represent new B. germanica-specific ESTs. Some genes which drew our attention are discussed. The information obtained increases our understanding of the B. germanica genome.
INTRODUCTION
The cockroach is among the oldest winged insects known, and its habits are closely associated with those of humans. Over four thousand species of cockroach are known, and about thirty species are harmful to humans in various ways. The importance of the German cockroach has been emphasized because it is the most populous and has the widest distribution (Ross and Cochran, 1975). Blattella germanica is also a well known cause of allergic diseases, rather than acting as a vector of infectious diseases (Richman et al., 1984).
Previous genetic studies on B. germanica have been limited to the study for some of its genes, e.g., allergen genes (Arruda et al 1995; Helm et al., 1996) and the cytochrome P450 gene, which is related with juvenile hormone or insecticide tolerance (Martinez-Gonzalez and Hegardt 1994; Scharf et al., 1998).
The generation and analysis of expressed sequence tags (ESTs) provides useful information on development, metabolism, virulence factors, drug targets, and pathogenesis in various organisms (el-Sayed et al., 1995; Wu et al., 1996; Manger et al., 1998; Bahl et al., 2003).
To understand more about the expression pattern of its genome, we generated ESTs from a cDNA library of B. germanica. The analysis of such data provides valuable insights into the metabolism and growth of German cockroach.
MATERIALS AND METHODS
Cockroach breeding
Adult male and female German cockroaches were reared at 24℃ on an artificial diet, and given free access to water.
cDNA library construction
A B. germanica cDNA library was constructed using Uni-ZAP™-XR expression vector (Stratagene, USA). In brief, total RNA was isolated from 3 g of the midgut of adult B. germanica. After phenol extraction and ethanol precipitation, poly(A+) RNA was purified using a Stratagene Poly(A) Quick mRNA Isolation Kit, in accordance with the manufacturer's instructions. First-strand cDNA synthesis of the isolated poly(A+) RNA was then conducted in 50 µl reaction volumes, using 50 units of MMLV-reverse transcriptase at 37℃ for 60 minutes. cDNA synthesis was primed using 5 µg oligo dT18. Second-strand synthesis was then conducted using RNase H and DNA polymerase I. After blunting the cDNA termini, EcoR I adaptor ligation and EcoR I phosphorylation were performed. Gel regions containing DNA molecules of length <400bp were then removed by Sepharose CL-2B gel filtration. Purified cDNA was ligated using dephosphorylated EcoR I Uni-ZAP™-XR vector arms, according to the manufacturer's instructions (Stratagene, USA), and then incubated using in vitro packaging extracts (Stratagene, USA).
Sequencing of randomly selected cDNA clones
Colonies from E. coli XL-1 Blue MRF cells harboring plasmid were obtained en masse by in vivo excision using assistant helper phage. Random recombinant clones were selected by blue-white color selection of colonies grown on LB-ampicillin agar plates. Plasmids containing the cDNA insert were extracted using a Wizard plasmid DNA purification system (Promega, Madison, WI, USA), and the existence of cDNA inserts was confirmed by gel electrophoresis after double digestion with EcoR I and Xho I. cDNA inserts were sequenced at DNA Sequencing Service (Macrogen, Seoul, Korea).
Basic local alignment search tool (BLAST) search analysis
Sequence outputs were manually edited to remove vector and ambiguous sequences. Sequence outputs of <100 bases in length were also rejected. The sequence data of cDNA clones obtained by random partial sequencing were searched for using BLAST at the National Center for Biotechnology Information (NCBI) for similarities in nucleic acid and protein databases. The BLASTN algorithm was used in conjunction with a nucleotide sequence database with a probability (P) cut-off of 10-4. Matches of translational products versus nucleic acid sequences search for using the BLASTX algorithm with a probability (P) cut-off of 10-4. Scores >160 for BLASTN or >80 for BLASTX were considered significant.
RESULTS
The submitted ESTs for BLAST searching comprised 363 ESTs from 465 randomly selected clones of the cDNA library of B. germanica. Clones of <100 bp in length or not successfully sequenced were excluded (102 clones). The average size of the 363 ESTs was 604 bp. These 363 sequenced clones were divided into three groups based on matches with public data sequences (Table 1). The matched with database group of 154 clones, showed high homology with the DNA sequence of B. germanica or other organisms. Of these clones, 10 ESTs corresponded to 3 previously identified B. germanica genes, including cytochrome coxidase subunit I, cytochrome oxidase II, and major allergen Bla g 1. The non-matched to database group contained 80 clones. One hundred twenty nine clones weren't significant.
Composition and ESTs categories of Blattella germanica cDNA library
ESTs were classified into putative function categories based on BLAST search results with associated predicted or known functions (Table 2). The most frequently found gene was that of ribosomal protein as 31 clones (27%). Nineteen genes (18%) were of uncertain function. Twenty, 12 and 12 ESTs corresponded to energy metabolism, enzymes related to metabolism and others, and 4 ESTs to allergens, 7 to proteases, 3 to protease inhibitors, 4 to translation factors, and 1 to cell signaling pathway.
Database match of Blattella germanica EST to the genes of the other organisms
DISCUSSION
The ESTs of 363 clones from a randomly selected 464 clones of B. germanica cDNA library were submitted for BLAST search and analyzed to determine the transcribed genome sequences. One hundred fifty four matched ESTs showed high homology with genes of various organisms including B. germanica. The ESTs of 41 clones showed the redundancy to other ESTs. Ten clones that had shared exact homology with genes previously characterized in B. germanica corresponded to 3 genes, cytochrome oxidase subunit 1, cytochrome oxidase II, and major allergen Bla g 1. Sixty-five ESTs were confirmed from among the 80 not-matched clones.
The most abundant group of ESTs in this study belonged to the ribosomal proteins. This result was expected because ribosomal protein genes are expressed ubiquitously at all stages of development. Moreover, the ribosomal protein family is generally well conserved and contains about 55 proteins in prokaryotes and 88 in eukaryotes (Doudna and Rath, 2002). An increasing number of studies have reported that numerous ribosomal proteins have extra-ribosomal functions, such as, involvements with several human genetic disorders (Wool, 1996). A recent study reported that ribosomal protein promotes DNA base excision repair in mammals such as the human and the mouse. This protein gene was expected to be used to repair 8-oxoguanine in man (Cappelli et al., 2003). In addition, the ribosomal protein family provides valuable comparative genomic and phylogenic data on insecta (Landais et al., 2003).
Cytochrome ESTs followed ribosomal proteins in number. These included cytochrome b, cytochrome oxidase polypeptides I, II, and III, cytochrome oxidase subunits II, and III, and cytochrome P450. These abundances could be explained by the high mRNA expression levels of cytochrome c oxidase subunit 1 in gut and fat bodies (Martinez-Gonzalez and Hegardt, 1994). The cytochrome c oxidase subunit I and the cytochrome P450 genes are over-expressed in pyrethroid-resistant strains of B. germanica (Pridgeon and Liu, 2003). Moreover, the cytochrome oxidase and P450 genes are good targets for the control of insecticide resistant German cockroach. The complete nucleotide sequences of the mitochondrial genome of several insects were recently identified for several purposes, such as, medicinal, sanitational, and forensic (Bae et al., 2004; Kim et al., 2005). The complete cockroach mitochondrial genome will be a useful source of information for molecular and evolutionary studies and for cockroach control.
B. germanica have been reported to have n=11 or n=12 chromosomes (Cochran and Ross, 1967; Ock and Kim, 1989). Although no specific information is available on the genome size of the German cockroach, it has been estimated to be ca. 1 × 1010 bp and CV=2.0 (haploid c-value in pg) (Ussery and Hallin, 2004), which is three times as large as the human genome. Wen et al (2001) reported that the B. germanica P450 gene is related to five pseudogenes compared to two pseudogenes in Drosophila. These pseudogenes, especially nuclear mitochondrial pseudogenes, have recently been viewed as tools for clarifying the relationship between DNA loss and genome size. Bensasson et al., (2001) reported that rates of DNA loss in pseudogenes are slow in the mountain grasshopper. However, in Drosophila, rates were high enough to contribute to the paucity of pseudogene sequences in the genome. The presence of many copies of pseudogenes is likely to explain the large genome size of B. germanica.
Of the protease genes identified in this study, trypsin influences growth and metamorphosis. Aalberse (2000) classified about 40 allergens into 4 structural families and other structures and designated trypsin-like serine proteases as one group of the antiparallel β-strands family. Moreover, there are reports that proteases extracted from B. germanica may have allergenic properties (Iraneta et al., 1999; Wongtim et al., 1993). We confirmed in a previous study that the trypsin of B. germanica reacts with the sera of allergic patients (Ock et al., 2005). A further characterization of trypsin in this respect would provide information of allergy, since trypsin plays an important part in the activation of PAR-2 (protease-activated receptor-2).
The clone Bg9033 was identified as a lysozyme. The secretion of lysozymes is known to be increased in the gastrointestinal tract of B. germanica during metamorphosis and food ingestion (Aigaki et al., 2002). Thus genetic information on lysosome would be helpful in studies of cockroach metamorphosis and digestion. The clone Bg6014, a catalase is known to affect defense mechanism and to expand life span by blocking free hydroxyl radical production in Drosophila (Hotokezaka et al., 2002; Missirlis et al., 2001). Further studies on catalase, cytochrome oxidase, and P450 would provide information useful for cockroach control.
In addition, elongation factors, ubiquitin, iron storage protein ferritin, and G protein-coupled receptor were all confirmed to be present in the German cockroach.
In the present study, we found 363 cDNA clones in the German cockroach genome, and 360 of these were identified for the first time in the German cockroach. These ESTs should provide valuable information on the development and metabolism of B. germanica and lead to the discovery of control targets.