Expressed sequence tags analysis of <italic>Blattella germanica</italic>

Hyang Suk Chung; Tai Hyun Yu; Bong Jin Kim; Sun Mi Kim; Joo Yeong Kim; Hak Sun Yu; Hae Jin Jeong; Mee Sun Ock

doi:10.3347/kjp.2005.43.4.149

Korean J Parasito > Volume 43(4):2005 > Article

Chung, Yu, Kim, Kim, Kim, Yu, Jeong, and Ock: Expressed sequence tags analysis of Blattella germanica

Original Article

The Korean Journal of Parasitology 2005; 43(4): 149-156.

Published online: December 20, 2005

DOI: https://doi.org/10.3347/kjp.2005.43.4.149

Expressed sequence tags analysis of Blattella germanica

Hyang Suk Chung¹, Tai Hyun Yu¹, Bong Jin Kim², Sun Mi Kim², Joo Yeong Kim², Hak Sun Yu³, Hae Jin Jeong³, Mee Sun Ock²

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 August 19, 2005 Accepted November 08, 2005

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.

Key words: Blattella germanica, cDNA library, ESTs, BLAST search, novel genes

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.

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.

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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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