Expressed sequence tags analysis of Blattella germanica

Article information

Korean J Parasitol. 2005;43(4):149-156
Publication date (electronic) : 2005 December 20
doi : https://doi.org/10.3347/kjp.2005.43.4.149
1Department of Otorhinolaryngology, Kosin University College of Medicine, Busan 602-703, Korea.
2Department of Parasitology, Kosin University College of Medicine, Busan 602-703, Korea.
3Department of Parasitology, Pusan National University College of Medicine, Busan, 602-739, Korea.
Corresponding author (sunnyock@kosin.ac.kr)
Received 2005 August 19; Accepted 2005 November 08.

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.

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Article information Continued

Table 1.

Composition and ESTs categories of Blattella germanica cDNA library

Group No. of clones
Total clones sequenced 465
 ESTs submitted to dbEST database 363
 Match to database 154
  Clone with homology to B. germanica (redundant clones 7) 10
  Clone with homology to other organisms (redundant clones 34) 144
 Non-match to database 80
 Non-significant clones 129

Table 2.

Database match of Blattella germanica EST to the genes of the other organisms

Clone No Length Accession No Putative homologue Organism Score P(N)
Ribosomal protein
 Bg11019 831 NP_524726 ribosomal protein L8 Drosophila melanogaster 83 4.00E-15
 Bg9035 659 AAL26575 ribosomal protein L8 Spodoptera frugiperda 311 3.00E-84
 Bg9065 644 AAK76989 ribosomal protein L9 Spodoptera frugiperda 327 4.00E-89
 Bg3103 381 AAK83857 ribosomal protein L17/23 Spodoptera frugiperda 154 2.00E-37
 Bg10031 863 AAL62470 ribosomal protein L18A Spodoptera frugiperda 259 3.00E-68
 Bg9046 286 AAL26577 ribosomal protein L29 Spodoptera frugiperda 108 1.00E-23
 Bg9031 443 CAC19413 ribosomal protein L31 Heliothis virescens 186 5.00E-47
 Bg11023 504 AAK92169 ribosomal protein L35A Spodoptera frugiperda 163 8.00E-40
 Bg9025 354 AAK92172 ribosomal protein L37A Spodoptera frugiperda 155 8.00E-38
 Bg12004 520 NP_476874 ribosomal protein S2 Drosophila melanogaster 226 1.00E-58
 Bg11037 829 AAL26579 ribosomal protein S3A Spodoptera frugiperda 348 5.00E-95
 Bg9014 562 NP_524884 ribosomal protein S14 Drosophila melanogaster 212 2.00E-54
 Bg11059 951 AAK92190 ribosomal protein S21 Spodoptera frugiperda 57 2.00E-07
 Bg7009 357 P47991 60S ribosomal protein L6 Caenorhabditis elegans 73 1.00E-13
 Bg9060 295 P32429 60S ribosomal protein L7A Gallus gallus 75 2.00E-13
 Bg8043 937 O96647 60S ribosomal protein L10 Bombyx mandarina 131 2.00E-31
 Bg4022 563 P46222 60S ribosomal protein L11 Drosophila melanogaster 303 2.00E-82
 Bg8048 407 P41126 60S ribosomal protein L13 Drosophila melanogaster 119 1.00E-27
 Bg8031 619 P41093 60S ribosomal protein L18A Drosophila melanogaster 249 7.00E-72
 Bg6016 832 P36241 60S ribosomal protein L19 Drosophila melanogaster 164 2.00E-40
 Bg4018 504 P23131 60S ribosomal protein L23 Homo Sapiens 215 3.00E-56
 Bg8025 550 Q02877 60S ribosomal protein L26 Homo Sapiens 133 2.00E-31
 Bg7021 505 P46615 60S ribosomal protein L32 Drosophila pseudoobscura 234 7.00E-62
 Bg7041 514 P02433 60S ribosomal protein L32 Homo Sapiens 167 8.00E-42
 Bg9006 888 AAK921 60S ribosomal protein L35 Spodoptera frugiperda 114 1.00E-24
 Bg8001 352 Q962S7 60S ribosomal protein L37 Spodoptera frugiperda 114 4.00E-26
 Bg8015 572 P05389 60S acidic ribosomal protein P2 Drosophila melanogaster 94 1.00E-19
 Bg5006 819 P52813 40S ribosomal protein S3A Anopheles gambiae 268 7.00E-81
 Bg9071 221 P55830 40S ribosomal protein S3A Drosophila melanogaster 64 3.00E-10
 Bg10029 947 P02350 40S ribosomal protein S3A Xenopus laevis 147 3.00E-36
 Bg11001 890 P47835 40S ribosomal protein S3B Xenopus laevis 285 7.00E-79
Energy metabolism
 Bg7011 871 P33502 NADH-Ubiquinone oxidoreductase chain 1 Anopheles quadrimaculatus 273 4.00E-73
 Bg8035 848 P29867 NADH-Ubiquinone oxidoreductase chain 2 Drosophila mauritiana 119 6.00E-27
 Bg9045 899 Q34048 NADH-Ubiquinone oxidoreductase chain 4 Ceratitis capitata 248 5.00E-65
 Bg7025 510 Q34050 NADH-Ubiquinone oxidoreductase chain 6 Ceratitis capitata 112 4.00E-25
 Bg1003 564 P07704 cytochrome b Drosophila yakuba 241 4.00E-64
 Bg9056 836 AAG17094 cytochrome b Bifiditermes improbus 217 2.00E-81
 Bg10006 942 AAG17097 cytochrome b Cryptotermes cynocephalus 230 1.00E-59
 Bg6013 905 P00400 cytochrome c oxidase polypeptide I Drosophila yakuba 207 1.00E-53
 Bg7003 219 P00399 cytochrome c oxidase polypeptide I Drosophila melanogaster 51 5.00E-07
 Bg8054 123 P50671 cytochrome c oxidase polypeptide I Choristoneura rosaceana 42 2.00E-04
 Bg5016 695 P29877 cytochrome c oxidase polypeptide II Periplaneta americana 269 3.00E-72
 Bg8064 150 P98048 cytochrome c oxidase polypeptide II Yponomeuta malinellus 62 2.00E-10
 Bg8068 379 P29877 cytochrome c oxidase polypeptide II American cockroach 173 5.00E-44
 Bg4013 783 P14574 cytochrome c oxidase polypeptide III Locusta migratoria 223 4.00E-58
 Bg8045 883 P00417 cytochrome c oxidase polypeptide III Drosophila melanogaster 221 1.00E-57
 Bg12010 983 AAB31450 cytochrome c oxidase subunit I Blattella germanica 203 3.00E-51
 Bg3202 387 AAF89137 cytochrome oxidase subunit III Cicindela belfragei 152 9.00E-37
 Bg10013 740 AAG01168 cytochrome oxidase subunit III Samia cynthia ricini 229 2.00E-59
 Bg3209 430 BAA32127 cytochrome oxidase II Blattella germanica 244 9.00E-65
 Bg7024 795 Q9V4U9 cytochrome P450 6a13 Drosophila melanogaster 120 3.00E-27
Allergen
 Bg8050 287 AAB82404 Cr-PII Periplaneta americana 58 2.00E-08
 Bg10001 294 AAC34737 Cr-PII allergen Periplaneta americana 58 2.00E-08
 Bg1010 340 AAD13530 major allergen Blag1.0101 Blattella germanica 121 2.00E-27
 Bg7008 365 AAD13532 major allergen Blag1.0101 Blattella germanica 135 1.00E-31
Protease
 Bg6009 408 P35035 Trypsin 1 precursor Anopheles gambiae 123 1.00E-28
 Bg3106 241 P35036 Trypsin 2 precursor Anopheles gambiae 87 3.00E-17
 Bg4101 439 S35339 trypsin (EC 3.4.21.4) 1 precursor Anopheles gambiae 123 4.00E-28
 Bg11002 384 AAD31269 trypsinogen Rdo T3 precursor Rhyzopertha dominica 130 2.00E-30
 Bg3109 382 P04069 Carboxypeptidase B Astacus astacus 84 3.00E-16
 Bg11072 430 1EQ9A Chain A, Crystal Structure Of Fire Ant Chymotrypsin Solenopsis invicta 86 7.00E-17
 Bg11049 534 AAA97479 Astryp1 Anopheles stephensi 129 2.00E-29
Enzyme related to metabolism
 Bg4008 607 Q9Y600 Cysteine sulfinic acid decarboxylase Homo sapiens 57 3.00E-08
 Bg6014 838 Q59296 Catalase Campylobacter jejuni 67 3.00E-11
 Bg8004 757 P26221 Endoglucanase E-4 precursor Thermobi fidafusca 134 1.00E-31
 Bg9010 847 S41881 alpha-amylase (EC 3.2.1.1) 1 precursor Litopenaeus vannamei 192 4.00E-48
 Bg9051 607 BAB91145 beta-glucosidase Neotermes koshunensis 82 3.00E-15
 Bg6015 442 P49010 beta-N-acetylglucosaminidase precursor Bombyx mori 120 8.00E-28
 Bg9015 949 P18173 Glucosedehydrogenase Drosophila melanogaster 100 2.00E-20
 Bg9043 589 JC4081 surcease/fructanase precursor Actinomyces naeslundii 52 3.00E-06
 Bg11053 924 AAC79122 alpha-amylase Drosophila ananassae 195 6.00E-49
 Bg10043 869 A34406 aldehydereductase (EC 1.1.1.21) Oryctolagus cuniculus 122 6.00E-27
 Bg9033 835 AAB61345 lysozyme Anopheles darlingi 69 6.00E-11
 Bg10002 453 BAB33297 Esterase-like protein (ESR-LP) Bombyx mori 67 8.00E-11
Protease inhibitor
 Bg5047 340 Q06684 Rhodniin (Thrombin inhibitor) Rhodnius prolixus 58 3.00E-09
 Bg9028 404 S45677 proteinase inhibitor Pacifastacus leniusculus 44 4.00E-04
 Bg10033 1026 AAK57342 thrombin inhibitor infestin precursor Triatoma infestans 64 2.00E-09
Translation
 Bg9029 888 NP_524611 elongation factor 1 alpha 100E Drosophila melanogaster 233 3.00E-60
 Bg9007 377 P29522 elongation factor 1-β Bombyx mori 66 5.00E-41
 Bg12018 758 BAB21109 elongation factor 1 delta Bombyx mori 130 1.00E-29
 Bg5012 486 Q9VL18 elongation factor 1-delta Drosophila melanogaster 67 2.00E-11
Cell signaling pathway
 Bg11067 384 Q09966 Putative G protein-coupled receptor B0244.7 Caenorhabditis elegans 30 3.9
Others
 Bg8028 901 P14792 Ubiquitin Caenorhabditis elegans 109 5.00E-14
 Bg10044 322 NP_476776 Ubiquitin fusion 52 Drosophila melanogaster 140 3.00E-33
 Bg5014 484 P22943 12kDa heat shock protein Saccharomy cerevisiae 94 1.00E-19
 Bg7040 829 P41822 ferritin subunit precursor Aedes aegypti 82 8.00E-16
 Bg9050 583 NP_523683 Peroxiredoxin2540 Drosophila melanogaster 84 2.00E-19
 Bg7005 492 O43653 Prostate stem cell antigen precursor Homo Sapiens 42 5.00E-04
 Bg4010 359 P40618 High mobility group protein 4 (HMG-4) Gallus gallus 55 3.00E-08
 Bg3208 433 AAH10444 matrilin2 Homo Sapiens 49 1.00E-05
 Bg8012 395 AAM21357 mucin-like protein 1 Ctenocephalides felis 49 8.00E-06
 Bg8016 842 O76767 ER lumen protein retaining receptor Drosophila melanogaster 140 3.00E-33
 Bg8039 447 Q27377 odorant-binding protein A10 precursor Drosophila melanogaster 5 1.00E-06
 Bg9016 880 AAA51540 4F2 antigen heavy chain Homo sapiens 71 1.00E-11
Not classified
 Bg9041 645 AAF45949 CG3556 gene product Drosophila melanogaster 85 5.00E-16
 Bg9040 401 NP_611703 CG4250 gene product Drosophila melanogaster 63 7.00E-10
 Bg11018 927 AAF55754 CG4362 gene product Drosophila melanogaster 84 3.00E-15
 Bg9020 902 NP_611243 CG6459 gene product Drosophila melanogaster 133 3.00E-30
 Bg9037 304 AAF50709 CG6592 gene product Drosophila melanogaster 65 1.00E-10
 Bg10012 854 NP_612081 CG9119 gene product Drosophila melanogaster 90 2.00E-19
 Bg9069 425 AAF48872 CG6696 gene product Drosophila melanogaster 77 3.00E-14
 Bg9044 833 AAF56428 CG10423 gene product Drosophila melanogaster 118 9.00E-26
 Bg7001 899 AAF58797 CG12405 gene product Drosophila melanogaster 100 3.00E-20
 Bg8018 285 P30652 23.7KD protein ZK6436 in chromosome III Caenorhabditis elegans 51 4.00E-07
 Bg9003 484 A45835 Ly6 homolog RK10 precursor Norway rat 47 8.00E-05
 Bg9055 877 AAF91388 SocE Myxococcus xanthus 99 4.00E-20
 Bg11070 856 NP_523610 clumsy Drosophila melanogaster 123 2.00E-34
 Bg11054 864 NP_476631 RpL19-P1;Enhancer of Delta KP135 Drosophila melanogaster 143 2.00E-33
 Bg12020 226 E81737 hypothetical protein TC0128 Chlamydia muridarum 44 4.00E-04
 Bg4020 841 P34472 136.3kD a protein F58A4.5 in chromosome III Caenorhabditis elegans 76 6.00E-14
 Bg9061 245 AAL49280 RE74144p Drosophila melanogaster 44 5.00E-04
 Bg10005 634 NP_502360 Arabidopsis pathogenesis-related protein 5 like Caenorhabditis elegans 109 2.00E-23
 Bg11011 547 AAL31950 CDH1-D Gallus gallus 51 7.00E-14