Complete genome sequence of Serratia ureilytica KML.E1, a copper-tolerant strain isolated from a disused tungsten mine.

Serratia ureilytica KML.E1 was recovered from a disused tungsten mine in Hong Kong and can tolerate copper(II) concentrations up to 90 mM. Its complete genome, a single chromosome of 5,094,661 bp (59.68% G+ C), was established through hybrid assembly.

A DNA-binding protein, tfp1, involved in juvenile hormone-regulated gene expression in Locusta migratoria.

A partially palindromic 15-nt. sequence upstream from a juvenile hormone-regulated gene (jhp21) was previously identified in the African migratory locust, Locusta migratoria. This sequence was proposed as a juvenile hormone (JH) response element (JHRE), and a protein that bound to it, as a transcription factor (TF). A yeast strain was constructed containing four tandem copies of the JHRE and after transfection with a cDNA library made to fat bodies from vitellogenic females, yeast one-hybrid experiments yielded sequences for four putative binding proteins. One of these sequences, corresponding to a transcript that was present in fat body irrespective of JH stimulation, encodes a 35kDa protein. This was designated tfp1 and appears to have a leucine zipper motif and a lipid-binding motif. Recombinant tfp1 bound to JHRE in electrophoretic mobility shift experiments and addition of tfp1 antibody in the binding reaction resulted in the disappearance or shift of TF. We suggest that JH induces the association of pre-existing proteins, including tfp1, to form an active complex, which binds to the JHRE upstream from jhp21 and regulates its transcription.

A locust DNA-binding protein involved in gene regulation by juvenile hormone.

Although juvenile hormone (JH) has essential roles in insect development and reproduction, the molecular mechanisms of gene regulation by JH remain an enigma. In Locusta migratoria, the partially palindromic 15-nt sequence, GAGGTTCGAG(A)/(T)CCT(T)/(C), found upstream of a JH-induced gene, jhp21, was designated as a putative juvenile hormone response element (JHRE). When JH-deprived adult female locusts were treated with the active JH analog, methoprene, a fat body nuclear factor that bound specifically to JHRE appeared after 24 h. Binding exhibited a preference for an inverted repeat with GAGGTTC in the left half-site, a single nucleotide spacer, and a right half-site in which some variation is acceptable. Binding to JHRE was abolished by phosphorylation catalyzed by a C-type protein kinase present in the nuclear extracts. The DNA-binding protein is thus believed to be a transcription factor, which is brought to an active state through the action of JH and then participates in the regulation of certain JH-dependent genes.

Sequences of elongation factors-1 alpha and -1 gamma and stimulation by juvenile hormone in Locusta migratoria.

Two cDNAs encoding the alpha and gamma subunits of translation elongation factor-1 (EF-1) have been cloned and sequenced from the African migratory locust, Locusta migratoria. Southern blotting and real-time PCR analyses indicated that these sequences represent single copy genes. Comparison with sequences from other species indicated greater conservation for EF-1 alpha than for EF-1 gamma. The developmental profiles for EF-1 alpha and -1 gamma mRNA expression in the fat body paralleled reported changes in the hemolymph juvenile hormone (JH) titer in the fifth instar and were elevated during early reproductive maturation in the female adult. In maturing adults, there was a greater accumulation of EF-1 alpha and -1 gamma transcripts in females than in males. The levels of both transcripts were greatly increased by an enriched diet, previously shown to elevate JH titers and accelerate vitellogenin production. Treating JH-deprived adult females with the JH analog, methoprene, resulted in more than doubling of transcript levels of both genes, supporting the hypothesis that JH could stimulate the accumulation of LmEF-1 alpha and -1 gamma transcripts. We suggest that production of elongation factors, increased by JH, may contribute to the massive protein synthesis required for egg production.

Cloning and characterization of an ecdysone receptor cDNA from Locusta migratoria.

To facilitate studies on the hormonal control of development in the migratory locust, Locusta migratoria, we have undertaken the cloning of cDNAs for nuclear hormone receptors. Sequences obtained by polymerase chain reaction (PCR) showed homology with receptor family members including the ecdysteroid receptor (EcR). A cDNA clone corresponding to the EcR fragment includes an open reading frame of 1622 nucleotides, predicting a 59 kDa protein showing clear homology with EcRs and distinct from other classes of nuclear receptors. Northern analysis revealed a major transcript of 9.2 kb. In fifth instar fat body, the transcript was most abundant at the end of the instar when ecdysone titres are highest. There was no obvious evidence of EcR regulation by a juvenile hormone analog. Although its role in development may be similar, the locust ecdysone receptor (LmEcR) is divergent from EcRs characterized from insects belonging to the dipteran and lepidopteran orders, presumably reflecting the more ancestral sequence in the relatively primitive locust.

Sequence of the hexameric juvenile hormone-binding protein from the hemolymph of Locusta migratoria.

The cDNA for the hexameric hemolymph juvenile hormone-binding protein (JHBP) from the migratory locust has been cloned and sequenced. Antiserum raised against purified JHBP was used to identify clones in an expression library. The 4.3-kilobase JHBP mRNA codes for 668 amino acids (74.4 kDa) and contains 2 kilobases of 3'-untranslated region. The derived amino acid sequence reveals that locust JHBP represents a new group within the hexamerin family of arthropod proteins. JHBP appears to be more closely related to arthropod hemocyanins, the believed ancestors of the family, than to the other known insect hexamerins. The mRNA shows a high (89%) bias to codons ending in G or C and the codons ending in A or T are clustered and concentrated toward the 5' end, suggesting a mosaic gene structure. The recombinant bacterially expressed protein bound [3H]JH III with the same affinity as the protein from hemolymph. A truncated version of JHBP lacking 53 amino acids from the N terminus did not bind JH III. Hybridization analysis of fat body JHBP mRNA in locusts that had been treated with precocene and a JH analog did not give clear evidence for regulation by JH.

Cloning and upstream sequence of a juvenile hormone-regulated gene from the migratory locust.

As a step toward analyzing the molecular mechanism of action of juvenile hormone (JH), the gene encoding a JH-inducible 21-kDa protein (Jhp21) produced in the fat body of the migratory locust has been cloned. Four exons, representing 750 nucleotides of cDNA sequence, were found to be distributed through 13 kb of genomic DNA. Upstream 2 kb of DNA has been sequenced and three potential hormone-response elements have been identified.

Juvenile hormone regulation of an insect gene: a specific transcription factor and a DNA response element.

We have used locust fat body nuclear protein extracts and upstream DNA of the juvenile hormone (JH)-inducible locust gene, jhp21, to examine the regulation of specific transcription by JH. Promoter activity was assayed with G-free cassette reporter constructs. Nuclear extracts from adult female fat body, previously exposed to JH or an analog, actively transcribe from the jhp21 promoter and a control adenovirus major late (AdML) promoter, whereas extracts from JH-deprived female fat body, or other tissues, transcribe strongly from the AdML promoter but weakly or not at all from the jhp21 promoter. Transcription is enhanced by sequences between -140 and -211 nt from the jhp21 transcription start point (tsp), which include a CAAT box, and also by sequences between -1056 and -1200. A 15-nt partially palindromic sequence element found at -1152, resembling known hormone response elements, was shown to stimulate transcription when restored to truncated jhp21 DNA. Two very similar sequences occur further upstream. In electrophoretic mobility shift assays (EMSA), the same sequence element was shown to specifically bind a protein that was present in nuclear extracts from JH-exposed, but not from JH-deprived, fat body. Several lines of evidence suggest that the DNA element may be a JH response element (JHRE). The JH-induced protein that binds to it appears to be a transcription factor that activates the initiation of JH target gene (jhp21) transcription, and could be a JH receptor.

The use of a juvenile hormone binding protein for the quantitative assay of juvenile hormone.

The suitability of the haemolymph juvenile hormone binding protein (JHBP) of Locusta migratoria for use in a competition assay for juvenile hormone (JH) III has been investigated, and a simple quantitative assay procedure using this protein has been developed. JHBP partially purified from haemolymph of precocene treated adult locusts gives rapid and stable binding of [3H]10R-JH III, and can be separated from the unbound hormone with hydroxylapatite (HAP). The sensitivity of the method is such that 0.15 pmol (40 pg) 10R-JH III gives 50% displacement of [3H]10R-JH III from the binding protein. Competition by JH II is about 5 times less and JH I about 10 times less than that by JH III, JH III diol and acid compete at least 1000 times less strongly. A procedure for extraction and assay of JH from 50 microliters haemolymph samples is described, the interference by non-specific haemolymph components is shown to be relatively small, and some data on JH III titres in maturing adult locusts are presented.

Roles of juvenile hormone, a brain factor and adipokinetic hormone in regulation of vitellogenin biosynthesis in locusta migratoria.

In maturing adult female migratory locusts, the rises in JH and Vg in the hemolymph are greatly accelerated by enriched feeding; hereafter the JH titer fluctuates with vitellogenic cycles, falling to a low level at the oviposition stage. In fat bodies incubated in vitro, the JH analog, methoprene, and brain extract from well-fed locusts (but not starved locusts) stimulated Vg synthesis synergistically. Repeated washing of fat bodies from oviposition stage locusts led to a rise in Vg synthesis after 4 h, which was prevented by addition of locust adipokinetic hormone (AKH). We conclude that at least three hormonal factors interact in the control of Vg synthesis in locus fat body: JH and a brain factor stimulate, reflecting development and nutrition, while AKH inhibits at the oviposition stage.

Properties and sequence of a female-specific, juvenile hormone-induced protein from locust hemolymph.

In the fat body of Locusta migratoria, an RNA transcript of about 800 nucleotides has been detected that is specific to the adult female and dependent on induction by juvenile hormone (JH) or an analog. The corresponding cDNA has been cloned (lambda 21) and a 718-base pair sequence determined. It encodes a 196-amino acid polypeptide, including a signal peptide. An NH2-terminal sequence has 24 out of 28 amino acids identical with those of a previously described 19K locust hemolymph protein, but the remainder of the sequence shows no similarity. From adult female hemolymph, a 21-kDa protein, designated 21K protein, has been purified, with an NH2-terminal sequence exactly matching that deduced from clone lambda 21. This 21K protein is found only in the adult female, is dependent on induction by JH, and is assumed to represent the product of the lambda 21 gene. It shows no immunochemical cross-reaction with locust 19K protein, apolipophorin III, nor with vitellogenin (Vg). Its isoelectric point is pH 5.4; it contains some carbohydrate. 21K protein is synthesized in adult female fat body, accumulates in hemolymph, and is taken up into the developing oocytes in parallel with Vg. In locusts deprived of JH with precocene, production of 21K protein and of lambda 21-hybridizing transcripts is induced by the JH analog, methoprene, in parallel with Vg and its mRNA. Because of its sex-, stage-, and JH-dependent regulation, coordinate with Vg, the 21K protein will be valuable for analysis of gene expression.

Modulation of DNA-binding proteins in Locusta migratoria in relation to juvenile hormone action.

Using a gel mobility shift assay, protein factors have been identified that may participate in the transcriptional regulation of the juvenile hormone (JH) induced vitellogenin A (VgA) gene of the migratory locust. Two DNA-binding proteins were found that were modulated in correlation with VgA gene activity during normal reproductive cycles and experimental stimulation by an active JH analogue (pyriproxyfen). One of these, JH factor (JHF), may be a regulator of VgA and perhaps other JH-responsive genes. The second, mobile factor (MF), appears to have a more general role in cellular responses to external stimuli such as JH and wounding. JHF and MF may represent factors that act together with the JH receptor to regulate VgA gene transcription.

Yolk polypeptide gene expression in cultured Drosophila cells.

The transfer of chimaeric plasmids to Drosophila melanogaster cell lines has been examined as a system for investigation of the hormonal regulation of the genes coding for D. melanogaster yolk polypeptide 1 (YP1) and Locusta migratoria vitellogenin B (VgB). Constructs containing promoters and putative 5'-regulatory sequences from these genes, ligated to bacterial chloramphenicol acetyltransferase (CAT) coding DNA, were transfected into Drosophila Kc (Kc-H) and S3 cells, and transient expression of CAT was assayed. Activity was expressed both from the homologous promoter of pYP1CAT and from the heterologous locust promoter of pVgCAT at comparable levels. In S3 cells, with calcium phosphate-mediated transfer of pYP1CAT there was a twofold induction of CAT activity after the addition of 10(-6) M ecdysterone, but no hormonal stimulation was noted when the polycation polybrene was used to achieve transfection. For Kc cells, calcium phosphate was ineffective for transfection, and after transfection with polybrene neither pYP1CAT nor pVgCAT was induced by the juvenile hormone (JH) analog methoprene. It is concluded that S3 cells may be useful for investigating the molecular basis of gene regulation by ecdysteroids, but conditions suitable for the analysis of JH action have not yet been established.

Distribution and sequence homogeneity of an abundant satellite DNA in the beetle, Tenebrio molitor.

The mealworm beetle, Tenebrio molitor, contains an unusually abundant and homogeneous satellite DNA which constitutes up to 60% of its genome. The satellite DNA is shown to be present in all of the chromosomes by in situ hybridization. 18 dimers of the repeat unit were cloned and sequenced. The consensus sequence is 142 nt long and lacks any internal repeat structure. Monomers of the sequence are very similar, showing on average a 2% divergence from the calculated consensus. Variant nucleotides are scattered randomly throughout the sequence although some variants are more common than others. Neighboring repeat units are no more alike than randomly chosen ones. The results suggest that some mechanism, perhaps gene conversion, is acting to maintain the homogeneity of the satellite DNA despite its abundance and distribution on all of the chromosomes.

Primary structure of apolipophorin-III from the migratory locust, Locusta migratoria. Potential amphipathic structures and molecular evolution of an insect apolipoprotein.

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Locusta migratoria, has been deduced from the sequence of its cloned cDNA. The mature hemolymph protein consists of 161 amino acids. Optimized alignments of this protein with apolipophorin-III from the tobacco hornworm, Manduca sexta, disclosed an overall sequence identity of only 29%, even though the two proteins are functionally equivalent. The L. migratoria sequence is composed of 12 repeating peptides that are variable in length. Six amphipathic helical segments of varying length were identified in each protein using a newly described algorithm for detecting such secondary structures. The degree of sequence identity between the two insect apoproteins is considerably less than that observed among orthologous mammalian apolipoproteins. However, calculation of the rates of synonymous and nonsynonymous nucleotide substitutions indicates that the insect genes may be evolving at rates similar to the mammalian apolipoprotein genes. Further comparative analyses of insect and mammalian apolipoproteins should provide insights about the limits of sequence diversity tolerated by their predicted amphipathic helical domains.

Vitellogenin mRNA in locust fat body: coordinate induction of two genes by a juvenile hormone analog.

Levels of vitellogenin (Vg) mRNA in Locusta migratoria fat body were determined as indicators of gene expression induced by the juvenile hormone analog methoprene. After injection of methoprene into juvenile hormone-deprived locusts, excised fat bodies were cultured with [3H]leucine for immunochemical assay of Vg synthesis, and RNA was assayed for Vg mRNA content by hybridization with probes from the previously cloned locust Vg genes A and B. In general, the rise in Vg mRNA paralleled the rise in Vg synthesis. During the primary response to methoprene (in female locusts in which the corpora allata had been destroyed immediately after emergence), Vg mRNA was first detected after 18-24 hr and accumulated rapidly between 36 and 48 hr. The secondary response (in locusts allatectomized during vitellogenesis and kept until Vg disappeared) was accelerated, as Vg mRNA was detectable at 12 hr and titers rose steeply after 18 hr. When Vg synthesis was prematurely induced by injection of methoprene into fifth-stage female larvae, the kinetics of mRNA accumulation were similar to those of primary stimulation in the adult. After allatectomy of vitellogenic females, fat body Vg mRNA decayed with a half-life of about 24 hr, roughly paralleling the decline in Vg synthesis. Assays with the two Vg probes showed coordinate accumulation of gene A and gene B messages under all conditions tested: during primary and secondary stimulation in adult females and in the low-level response obtained by treating male larvae with methoprene.

Cloning and 5' end nucleotide sequences of two juvenile hormone-inducible vitellogenin genes of the African migratory locust.

Screening of lambda libraries prepared with locust (Locusta migratoria) genomic DNA yielded overlapping clones containing two complete vitellogenin genes, designated A and B, along with extensive 3'- and 5'-flanking sequences. Genes A and B are about 12 kb and 10.5 kb long, respectively, and both hybridize on Northern blots with 6300-nucleotide RNAs previously identified as vitellogenin mRNAs. Each gene contains a large intron near the 5' end and must also contain other introns. Repetitive "Lm1 elements," similar to the human Alu repetitive DNA elements, and other repeated sequences occur within the large intron and in the 3'- and 5'-flanking regions of each gene. Southern blots showed no cross-hybridization between the major portions of mRNA-coding sequences of genes A and B. DNA sequences have been determined for the 5' terminal exons and flanking regions of both genes. Both genes contain short (56-58 nucleotide) 5' leader exons, which, unlike the rest of the coding region, are highly conserved in sequence (87% identical) and code for amino acids that resemble the amino-terminal sequences of vertebrate vitellogenins. The upstream 5'-flanking sequences of the two genes contain several short stretches of similarity that may be involved in juvenile hormone regulated expression.

The ribosomal RNA genes of Locusta migratoria: copy number and evidence for underreplication in a polyploid tissue.

From libraries of Locusta migratoria genomic DNA in bacteriophage lambda, clones have been isolated that hybridize with one or both of the 18S and 28S components of locust rRNA. Six clones studied show common patterns of restriction sites in the coding sequences and some include an intron in the 28S region. Subcloned sequences from within the 18S and 28S coding regions were used as probes in dot hybridization assays of the rRNA gene copy number in DNA prepared from several locust tissues and stages. The 18S and 28S probes gave similar results, showing about 4000 copies/haploid genome in testis, embryo, and fat body from larvae and immature adults of both sexes. This is by far the greatest rRNA gene copy number yet reported for any insect. DNA from reproductively mature adult female fat body showed a consistent reduction in the copy number by about one-third. Mature adult male fat body DNA showed no significant reduction in the copy number. It therefore appears that the final round of DNA replication in the adult female fat body, which produces 8-ploid and 16-ploid cells active in vitellogenin synthesis, involves underreplication of rDNA.

An ubiquitous interspersed DNA sequence family in an insect.

The genome of the African migratory locust, Locusta migratoria (Class Insecta, Order Orthoptera, Family Acrididae) contains an interspersed DNA sequence family, designated the Lm1 family. This family consists of short (approximately 195-bp), widely dispersed, highly reiterated (approximately 6 X 10(5) copies/haploid genome) repeat units, which account for about 2% of the locust genome. Lm1 repeats contain regions that closely resemble internal promoter sequences for RNA polymerase III, and they are structurally very similar to RNA polymerase III templates. Family members are flanked by short direct repeats, and are closely linked to structural genes. These features are reminiscent of the Alu family of man and other repeat sequence families, until now documented only in higher vertebrates.

Juvenile hormone binding by components of fat body cytosol from vitellogenic locusts.

Binding of 3H-labeled juvenile hormone (JH) to cytosol components of fat body from adult female Locusta migratoria, a tissue in which JH stimulates vitellogenin synthesis, has been characterized. Protein-bound JH is separated from unbound hormone with hydroxyapatite, which is found to provide a more sensitive and less variable assay than use of dextran-coated charcoal. By chromatography on DEAE-cellulose, three JH-binding components have been separated from cytosol. BP-I exhibited relatively stable binding with little degradation of the hormone, gave a Kd for JH-I by Scatchard analysis of 1.69 X 10(-8) M, and binding of JH-I was competed by JH-I and several synthetic JH analogs in an order corresponding to their JH activities. These characteristics suggest that BP-I may be a cytoplasmic JH receptor. BP-II, a minor component, also bound JH-I stably, but competition by analogs was not correlated with their hormone activity. BP-III caused rapid degradation of JH to JH acid and may be an esterase.