Genome size evaluations in cockroaches: New entries

In this paper, we report genome size (GS) values for nine cockroaches (order Blattodea, families Blattidae, Blaberidae and Ectobiidae, ex Blattelidae), three of which are original additions to the ten already present in the GS database: the death’s head roach (Blaberus craniifer), the Surinam cockroach (Pycnoscelus surinamensis) and the Madeira cockroach (Leucophaea maderae). Regarding the American cockroach (Periplaneta americana), the GS database contains two contrasting values (2.72 vs 3.41 pg); likely, the 2.72 pg value is the correct one as it is strikingly similar to our sperm DNA content evaluation (2.80 ± 0.11 pg). Also, we suggest halving the published GS of the Argentine cockroach Blaptica dubia and the spotted cockroach (the gray cockroach) Nauphoeta cinerea discussing i) the occurrence of a correlation between increasing 2n chromosome number and GS within the order Blattodea; and ii) the possible occurrence of a polyploidization phenomenon doubling a basic GS of 0.58 pg of some termite families (superfamily Blattoidea, epifamily Termitoidae).Key words: genome size, C-DNA content, cockroaches, Blattodea     

Conserved association of Argonaute 1 and 2 proteins with miRNA and siRNA pathways throughout insect evolution,from cockroaches to flies

Background

Argonaute proteins are key in RNA silencing. In Drosophila melanogaster, the five proteins of the Argonaute family participate in the pathways and mechanisms mediated by three types of small RNAs: piRNAs, miRNAs, and siRNAs. Two Argonaute proteins, Argonaute 1 (Ago1) and Argonaute 2 (Ago2), are associated with miRNA and siRNA mechanisms, which are the most thoroughly studied. The available data points to a sorting specialization of Ago1 for miRNAs and Ago2 for siRNAs. However, this has been demonstrated only in D. melanogaster, one of the most modified insects, which emerged some 100 million years ago. Thus, an important question is whether this association of Ago1 with miRNAs and Ago2 with siRNAs occurs generally in insects, or was a specific innovation in higher flies.

A Large Gene Cluster Encoding Several Magnetosome Proteins Is Conserved in Different Species of Magnetotactic Bacteria

In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization. We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium Magnetospirillum gryphiswaldense that could be assigned to two different genomic regions. Except for mamA, none of these genes have been previously reported to be related to magnetosome formation. Homologous genes were found in the genome sequences of M. magnetotacticum and magnetic coccus strain MC-1. The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD). A major gene cluster containing several magnetosome genes (including mamA and mamB) was found to be conserved in all three of the strains investigated. The mamAB cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation.     

Comparison of protein patterns in cockroach muscles innervated by fast or slow neurons

The protein composition of six of the coxal depressor muscles of the leg of the cockroach, Periplaneta americana, was analyzed by one and two dimensional SDS—polyacrylamide gel electrophoresis in conjunction with a sensitive silver stain. The proteins in each muscle were fractionated according to their solubilities in either low salt buffer, 1%NP-40, or 2%SDS. The gel patterns resulting from the electrophoresis of each of the fractions from “fast”, “slow”, and “mixed” type muscle were compared with reference to the innervation of the muscle by a particular motor neuron. More than 50 proteins were detected which were not common to all three types of muscle. These proteins were found to be distributed fairly equally amongst each of the three fractions. The large number of differentially expressed proteins observed, are not likely to be involved in specific neuromuscular recognition, but instead are probably correlated with the physiological state of the muscle.     

In vitro synthesis and release of proteins by fat body and ovarian tissue of Leucophaea maderae during the sexual cycle

Fat body, ovaries without their surrounding connective tissue, and ovarian connective tissue of the ovoviviparous cockroach Leucophaea maderae were incubated in vitro. Incorporation of ¹⁴C-labelled amino acids into proteins by all three tissues was investigated quantitatively and qualitatively during oöcyte maturation (corpora allata active), immediately after ovulation, and during the gestation period (corpora allata inactive). Proteins in the tissues and in the incubation media were processed separately. The qualitative analysis involving disk electrophoretic separation of Ringer soluble tissue proteins or of proteins in the incubation media and subsequent autoradiography of the dried gels showed that all three tissues synthesize the same 26 proteins in all stages of the sexual cycle. An additional fraction is produced by the ovarian connective tissue only. All three tissues synthesize proteins at a higher rate during oöcyte maturation than during gestation.     

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.     

Sucrose-binding lectin in regenerating cockroach (Periplaneta americana) legs: Its purification from adult hemolymph

Previously, we purified Periplaneta lectin from the hemolymph of adult Periplaneta americana (American cockroach) (Kubo and Natori Eur. J. Biochem. 168, 75–82, 1987). Immunoblotting analysis using antibody against Periplaneta lectin showed that the cockroach hemolymph contains another lectin that cross reacted immunologically with Periplaneta lectin. We have purified this new lectin (regenectin) to homogeneity. Affinity purified antibody against regenectin cross reacted with Periplaneta lectin. Thus, Periplaneta lectin and this new lectin were found to be different lectins sharing common antigenicity. After leg amputation, this new lectin was found to appear transiently at a specific stage of regeneration as revealed by immunoblotting, suggesting that it plays a role in the regeneration process.     

Specific activity of a Bacillus thuringiensis strain against Locusta migratoria manilensis

Bacillus thuringiensis (Bt) has played an important role in biocontrol of pests. However, insecticidal activity of B. thuringiensis against locusts has been rarely reported. Bt strain BTH-13 exhibiting specific activity to locusts was isolated from a soil sample in China and characterized. Its bipyramidal parasporal crystal is mainly composed of a protein of 129 kDa, and produces a mature toxin of 64 kDa after activation. The pattern of total DNA from BTH-13 showed a large and three small plasmid bands. Known δ-endotoxin genes, cry1Aa, cry1Ab, cry1Ac, cry1C, cry3, cry4 and cry7Aa were not found from strain BTH-13 by PCR amplification. The sequence analysis of a DNA fragment produced by PCR amplification with degenerate cry-selective primers revealed that the fragment encoded a δ-endotoxin segment, which exhibited some similarity to several Cry proteins (41% of the highest similarity to Cry7Ba1). Toxicity tests were performed against Locusta migratoria manilensis, and the results demonstrated that trypsin-treated sporulated cultures and crystal proteins had high toxicity to larval and adult locusts. Cry toxin of BTH-13 was detected on the midguts of treated locusts using immunofluorescent technology, which confirmed the site of action of the crystal proteins in their toxicity for locusts.     

Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.     

Bioprospecting for genes involved in the production of chitosanases and microcystin-like compounds in Anabaena strains

Bioinformatic tools guided PCR amplification assays were employed for analyzing two Anabaena strains A. laxa and A. iyengarii which exhibited chitosanase activity, allelopathic and fungicidal activity. Sequencing of a 297 bp fragment obtained by amplification with primers directed towards mcy A gene (involved in the production of microcystins), revealed significant similarity with the condensation domain, while amplification with specific primers towards N-methyltransferase (NMT) domain showed 59% similarity with a homologous domain in a toxic strain of Microcystis aeruginosa. An amplified product of 172 bp obtained using specific primers derived from the coding region of chitinase (chi IS) gene in Streptomyces sp., showed 100% similarity with hydrogenbyrinic acid a, c-diamide cobaltochelatase gene in Anabaena, and significant similarity with chi IS gene of Streptomyces sp. under less stringent conditions. The 663 bp sequence obtained by employing specific primers for chitosanase (choA) derived from Mitsuaria chitosanitabida 3001 strain, showed 100% similarity with glycoside hydrolase family three domain like protein(s). This study is a first time report on the presence of homologues of chitosanase in cyanobacteria which can play a role in allelopathic activity exhibited by these oxygenic photosynthetic prokaryotes.     

Complete Sequence of the Enterocin Q-Encoding Plasmid pCIZ2 from the Multiple Bacteriocin Producer Enterococcus faecium L50 and Genetic Characterization of Enterocin Q Production and Immunity

The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.     

Divergent polo box domains underpin the unique kinetoplastid kinetochore

Kinetochores are macromolecular machines that drive eukaryotic chromosome segregation by interacting with centromeric DNA and spindle microtubules. While most eukaryotes possess conventional kinetochore proteins, evolutionarily distant kinetoplastid species have unconventional kinetochore proteins, composed of at least 19 proteins (KKT1–19). Polo-like kinase (PLK) is not a structural kinetochore component in either system. Here, we report the identification of an additional kinetochore protein, KKT20, in Trypanosoma brucei. KKT20 has sequence similarity with KKT2 and KKT3 in the Cys-rich region, and all three proteins have weak but significant similarity to the polo box domain (PBD) of PLK. These divergent PBDs of KKT2 and KKT20 are sufficient for kinetochore localization in vivo. We propose that the ancestral PLK acquired a Cys-rich region and then underwent gene duplication events to give rise to three structural kinetochore proteins in kinetoplastids.     

A study of histones in amoeba nuclei by indirect immunofluorescence method.

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

Photoaffinity labeling and biochemical characterization of binding proteins for pheromones,juvenile hormones,and peptides

Radiolabeled photoaffinity analogs can be used to purify and characterize proteins involved in pheromone perception, juvenile hormone (JH) action, and neuropeptide reception. Several photoaffinity analogs and purification strategies are described for each of these physiological targets. First, a diazoacetate photoaffinity label is used to selectively modify the pheromone binding protein of the gypsy moth, Lymantria dispar. Reverse-phase HPLC is then employed to fractionate the male antennal proteins. Second, a tandem procedure involving preparative isoelectric focusing (IEF) and ion-exchange (IEX) HPLC is employed for the purification of the Manduca sexta hemolymph juvenile hormone binding protein (JHBP), which has now been cloned and sequenced. A separate application of this strategy for the purification of the 29 kDa JH I/methoprene receptor proteins from epidermal nuclei of M. sexta larvae is outlined. A new photolabel, farnesyl diazoketone, has been employed for the characterization of crustacean hemolymph methyl farnesoate binding proteins. Third, the development of neuropeptide photoaffinity labels is described for two systems: (1) the red pigment concentrating hormone (RPCH) of shrimp and (2) the allatostatins isolated from the brain of the cockroach Diploptera punctata.     

The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties

The methyl-CpG binding domain (MBD) family of proteins was defined based on sequence similarity in their DNA binding domains. In light of their high degree of conservation, it is of inherent interest to determine the genomic distribution of these proteins, and their associated co-repressor complexes. One potential determinant of specificity resides in differences in the intrinsic DNA binding properties of the various MBD proteins. In this report, we use a capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) and neutral capillaries to calculate MBD–DNA binding affinities. MBD proteins were assayed on pairs of methylated and unmethylated duplex oligos corresponding to the promoter regions of the BRCA1, MLH1, GSTP1 and p16^INK4a genes, and binding affinities for each case were calculated by Scatchard analyses. With the exception of mammalian MBD3 and Xenopus MBD3 LF, all the MBD proteins showed higher affinity for methylated DNA (in the nanomolar range) than for unmethylated DNA (in the micromolar range). Significant differences between MBD proteins in the affinity for methylated DNA were observed, ranging within two orders of magnitude. By mutational analysis of MBD3 and using CEMSA, we demonstrate the critical role of specific residues within the MBD in conferring selectivity for methylated DNA. Interestingly, the binding affinity of specific MBD proteins for methylated DNA fragments from naturally occurring sequences are affected by local methyl-CpG spacing.