Conceptual Translation Product Research Articles

Curiously composite structures of a retrotransposon and a complex repeat associated with chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

In Drosophila, telomere retrotransposons counterbalance the loss of telomeric DNA. The exceptional mechanism of telomere recovery characterized in Drosophila has not been found in lower dipterans (Nematocera). However, a retroelement resembling a telomere transposon and termed "RaTART" has been described in the nematoceran Rhynchosciara americana. In this work, DNA and protein sequence analyses, DNA cloning, and chromosomal localization of probes obtained either by PCR or by screening a genomic library were carried out in order to examine additional features of this retroelement. The analyses performed raise the possibility that RaTART represents a genomic clone composed of distinct repetitive elements, one of which is likely to be responsible for its apparent enrichment at chromosome ends. RaTART sequence in addition allowed to assess a novel subtelomeric region of R. americana chromosomes that was analyzed in this work after subcloning a DNA fragment from a phage insert. It contains a complex repeat that is located in the vicinity of simple and complex tandem repeats characterized previously. Quantification data suggest that the copy number of the repeat is significantly lower than that observed for the ribosomal DNA in the salivary gland of R. americana. A short insertion of the RaTART was identified in the cloned segment, which hybridized preferentially to subtelomeres. Like RaTART, it displays truncated sequences related to distinct retrotransposons, one of which has a conceptual translation product with significant identity with an endonuclease from a lepidopteran retrotransposon. The composite structure of this DNA stretch probably reflects mobile element activity in the subtelomeric region analyzed in this work.

The sea urchin (Strongylocentrotus purpuratus) test and spine proteomes.

BackgroundThe organic matrix of biominerals plays an important role in biomineral formation and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin, which is an important model organism for developmental biology and biomineralization, only few matrix components have been identified and characterized at the protein level. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of possible matrix proteins at the gene level, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy, proteomic analysis.ResultsWe identified 110 proteins as components of sea urchin test and spine organic matrix. Fourty of these proteins occurred in both compartments while others were unique to their respective compartment. More than 95% of the proteins were detected in sea urchin skeletal matrices for the first time. The most abundant protein in both matrices was the previously characterized spicule matrix protein SM50, but at least eight other members of this group, many of them only known as conceptual translation products previously, were identified by mass spectrometric sequence analysis of peptides derived from in vitro matrix degradation. The matrices also contained proteins implicated in biomineralization processes previously by inhibition studies using antibodies or specific enzyme inhibitors, such as matrix metalloproteases and members of the mesenchyme-specific MSP130 family. Other components were carbonic anhydrase, collagens, echinonectin, a α2-macroglobulin-like protein and several proteins containing scavenger receptor cysteine-rich domains. A few possible signal transduction pathway components, such as GTP-binding proteins, a semaphorin and a possible tyrosine kinase were also identified.ConclusionThis report presents the most comprehensive list of sea urchin skeletal matrix proteins available at present. The complex mixture of proteins identified in matrices of the sea urchin skeleton may reflect many different aspects of the mineralization process. Because LC-MS/MS-based methods directly measures peptides our results validate many predicted genes and confirm the existence of the corresponding proteins. Considering the many newly identified matrix proteins, this proteomic study may serve as a road map for the further exploration of biomineralization processes in an important model organism.

Characterization and expression of the Hex 110 gene encoding a glutamine‐rich hexamerin in the honey bee, Apis mellifera

An N-terminal amino acid sequence of a previously reported honey bee hexamerin, HEX 110 [Danty et al., Insect Biochem Mol Biol 28:387-397 (1998)], was used as reference to identify the predicted genomic sequence in a public GenBank database. In silico analysis revealed an ORF of 3,033 nucleotides that encompasses eight exons. The conceptual translation product is a glutamine-rich polypeptide with a predicted molecular mass of 112.2 kDa and pI of 6.43, which contains the conserved M and C hemocyanin domains. Semiquantitative and quantitative RT-PCR with specific primers allowed for an analysis of mRNA levels during worker bee development and under different physiological conditions. Concomitantly, the abundance of the respective polypeptide in the hemolymph was examined by SDS-PAGE. Hex 110 transcripts were found in high levels during the larval stages, then decreased gradually during the pupal stage, and increased again in adults. HEX 110 subunits were highly abundant in larval hemolymph, decreased at the spinning-stage, and remained at low levels in pupae and adults. In 5th instar larvae, neither starvation nor supplementation of larval food with royal jelly changed the Hex 110 transcript levels or the amounts of HEX 110 subunit in hemolymph. In adult workers, high levels of Hex 110 mRNA, but not of the respective subunit, were related to ovary activation, and also to the consumption of a pollen-rich diet.

Functional characterization of the promoter of the vitellogenin gene, AsVg1, of the malaria vector, Anopheles stephensi

Some genetic strategies for controlling transmission of mosquito-borne diseases call for the introgression of antipathogen effector genes into vector populations. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the expression of the effector molecules to maximize their efficacy. Vitellogenin (Vg)-encoding gene control sequences are candidates for driving tissue-, stage- and sex-specific expression of exogenous genes. One of the Anopheles stephensi Vg genes, AsVg1, was cloned and a full-length cDNA, as well as 850 base pairs adjacent to the 5′-end, were sequenced and characterized. Expression of AsVg1 is restricted to the fat body tissues of blood-fed females, and the amino acid sequence of the conceptual translation product is >85% identical to those of other anopheline Vgs. These characteristics support the conclusion that AsVg1 is a Vg-encoding gene. Functional analyses of the AsVg1 putative cis-regulatory sequences were performed using transgenic mosquitoes. The results showed that DNA fragments encompassing the 850 base pairs immediately adjacent to the 5′-end of the gene and the 3′-end untranslated region are sufficient to direct sex-, stage- and tissue-specific expression of a reporter gene. These data indicate that the AsVg1 promoter is a good candidate for controlling the expression of anti-pathogen effector molecules in this malaria vector mosquito.

Primary characterization and basal promoter activity of two hexamerin genes of Musca domestica

Hexamerins are high molecular-weight proteins found in the hemolymph of insects and have been proposed to function as storage proteins. In previous studies, two Musca domestica hexamerins, designated Hex-L and Hex-F were characterized. Hex-L is synthesized exclusively by the larval fat bodies, is secreted into the hemolymph and likely provides a source of amino acids and energy during metamorphosis. Hex-F synthesis is induced by a proteinaceous meal and occurs only in the adult insect fat bodies. Hex-F also is secreted into the hemolymph and it has been suggested that in females it may be an amino acid reservoir to be used during the final stages of egg formation. Genomic clones containing full-length copies of the genes MdHexL1 and MdHexF1, encoding subunits of the larval and the adult female hexamerin, respectively, were isolated. Complete nucleotide sequences, including the 5′-end untranscribed regions, were determined and analyzed for each of the genes. Comparisons of the conceptual translation products of the cloned genes indicated that MdHexL1 and MdHexF1 are related to the larval serum proteins (LSP) 1 and 2 of Calliphora vicina and Drosophila melanogaster. DNA fragments containing the putative promoters of the two hexamerin genes were compared and cloned into a plasmid vector so as to drive the expression of the GFP reporter gene. The constructs were assayed in vitro in transfected S2 Drosophila melanogaster cells demonstrating that the cloned M. domestica DNA fragments exhibit promoter activity.Hex L, Hex F Larval and female hexamerins respectively LSP Larval serum protein MdHexL1, MdHexF1 hexamerins of Musca domestica

Primary characterization and basal promoter activity of two hexamerin genes of Musca domestica

Hexamerins are high molecular-weight proteins found in the hemolymph of insects and have been proposed to function as storage proteins. In previous studies, two Musca domestica hexamerins, designated Hex-L and Hex-F were characterized. Hex-L is synthesized exclusively by the larval fat bodies, is secreted into the hemolymph and likely provides a source of amino acids and energy during metamorphosis. Hex-F synthesis is induced by a proteinaceous meal and occurs only in the adult insect fat bodies. Hex-F also is secreted into the hemolymph and it has been suggested that in females it may be an amino acid reservoir to be used during the final stages of egg formation. Genomic clones containing full-length copies of the genes MdHexL1 and MdHexF1, encoding subunits of the larval and the adult female hexamerin, respectively, were isolated. Complete nucleotide sequences, including the 5'-end untranscribed regions, were determined and analyzed for each of the genes. Comparisons of the conceptual translation products of the cloned genes indicated that MdHexL1 and MdHexF1 are related to the larval serum proteins (LSP) 1 and 2 of Calliphora vicina and Drosophila melanogaster. DNA fragments containing the putative promoters of the two hexamerin genes were compared and cloned into a plasmid vector so as to drive the expression of the GFP reporter gene. The constructs were assayed in vitro in transfected S2 Drosophila melanogaster cells demonstrating that the cloned M. domestica DNA fragments exhibit promoter activity.

The Musca domestica larval hexamerin is composed of multiple, similar polypeptides

The Musca domestica larval hexamerin (MdHex-L) is a hexameric glycoprotein with an apparent native molecular weight of 500 kDa. Seven different cDNAs that encode MdHex-L subunits were cloned and sequenced. Furthermore, amino acid sequences of isolated subunits were determined by the Edman degradation method and compared to the conceptual translation products derived from the cloned cDNAs. The obtained data indicate the existence of multiple forms of MdHex-L subunits and that these multiple forms may be grouped into three categories according to their percentages of nucleotide sequence identity.

Isolation of cDNA clones encoding putative odourant binding proteins from the antennae of the malaria-transmitting mosquito, Anopheles gambiae.

One way of controlling disease transmission by blood-feeding mosquitoes is to reduce the frequency of insect-host interaction, thus reducing the probability of parasite transmission and re-infection. A better understanding of the olfactory processes responsible for allowing mosquitoes to identify human hosts is required in order to develop methods that will interfere with host seeking. We have therefore initiated a molecular approach to isolate and characterize the genes and their products that are involved in the olfactory recognition pathway of the mosquito Anopheles gambiae, which is the main malaria vector in sub-Saharan Africa. We report here the isolation and preliminary characterization of several cDNAs from male and female A. gambiae antennal libraries that encode putative odourant binding proteins. Their conceptual translation products show extensive sequence similarity to known insect odourant binding proteins (OBPs)/pheromone binding proteins (PBPs), especially to those of D. melanogaster. The A. gambiae OBPs described here are expressed in the antennae of both genders, and some of the A. gambiae OBP genes are well conserved in other disease-transmitting mosquito species, such as Aedes aegypti and Culex quinquefasciatus.

Ovocalyxin-32, a Novel Chicken Eggshell Matrix Protein: ISOLATION, AMINO ACID SEQUENCING, CLONING, AND IMMUNOCYTOCHEMICAL LOCALIZATION

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate concomitantly with an organic matrix upon the eggshell membranes. Mineralization takes place in an acellular uterine fluid, which contains the ionic and matrix precursors of the eggshell. We have identified a novel 32-kDa protein, ovocalyxin-32, which is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. Sequencing of peptides derived from the purified protein allowed expressed sequence tag sequences to be identified that were assembled to yield a full-length composite sequence whose conceptual translation product contained the complete amino acid sequence of ovocalyxin-32. Data base searches revealed that ovocalyxin-32 has limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in the rat cerebral cortex and mast cells, and a skin protein, which is encoded by a retinoic acid receptor-responsive gene, TIG1. High level expression of ovocalyxin-32 was limited to the isthmus and uterus tissue, where immunocytochemistry at the light and electron microscope levels demonstrated that ovocalyxin-32 is secreted by surface epithelial cells. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell formation occurs.

Unusual molecular architecture of the Yersinia pestis cytotoxin YopM: a leucine-rich repeat protein with the shortest repeating unit

Many Gram-negative bacterial pathogens employ a contact-dependent (type III) secretion system to deliver effector proteins into the cytosol of animal or plant cells. Collectively, these effectors enable the bacteria to evade the immune response of the infected organism by modulating host-cell functions. YopM, a member of the leucine-rich repeat protein superfamily, is an effector produced by the bubonic plague bacterium, Yersinia pestis, that is essential for virulence. Here, we report crystal structures of YopM at 2.4 and 2.1 Å resolution. Among all leucine-rich repeat family members whose atomic coordinates have been reported, the repeating unit of YopM has the least canonical secondary structure. In both crystals, four YopM monomers form a hollow cylinder with an inner diameter of 35 Å. The domain that targets YopM for translocation into eukaryotic cells adopts a well-ordered, α-helical conformation that packs tightly against the proximal leucine-rich repeat module. A similar α-helical domain can be identified in virulence-associated leucine-rich repeat proteins produced by Salmonella typhimurium and Shigella flexneri, and in the conceptual translation products of several open reading frames in Y. pestis.

Tentative identification of a resilin gene in Drosophila melanogaster

A search of the Drosophila genome for gene products with similarities to the amino acid sequences of three tryptic peptides from locust ( Schistocerca gregaria) resilin gave two positive results: gene products CG15920 and CG9036. In both conceptual translation products a 62-residue region is present, which is identical to the resilin peptides in 29 positions. Gene product CG15920 has an amino acid composition closely resembling that of resilins from various insect species, and it has an N-terminal signal peptide sequence indicating that it is an extracellular protein. The 62-residue region shows similarity to the RR-2 sequence, which is common for a number of matrix proteins from insect solid cuticle. The N- and C-terminal regions flanking the 62-residue in CG15920 are dominated by 18 repeats of a 15-residue sequence and 11 repeats of a 13-residue sequence, respectively. The structures of the repeats predict that the peptide chain will fold in an irregular, extended beta-spiral, resembling the structures suggested for mammalian elastin and spider flagelliform silk, two materials which, like resilin, possess long-range elasticity. Accordingly, we suggest that gene product CG15920 is a Drosophila resilin precursor.

Characterization and expression analysis of two human septin genes , PNUTL1 and PNUTL2

The presence and role of septin proteins in yeast is well documented, but there is a growing appreciation for this family of proteins beyond yeast and extending to human cells. In this report we present the characterization and comparison of two highly similar human septin genes, PNUTL1 and PNUTL2. We compare the exon/intron structure of both genes, the steady-state mRNA levels in tumor cell lines and adult organs, the conceptual translation products from alternatively processed mRNAs and the development of specific immunologic reagents distinguishing either PNUTL1 or PNUTL2. The results illustrate a remarkable similarity between the two genes and their protein products while identifying specific differences in mRNA expression patterns. A summary of the described functional roles for mammalian septins is discussed along with an attempt to assimilate the alternative nomenclature existing for the same human septins, such as references to PNUTL1 and PNUTL2 as hCDCrel-1 and hCDCrel-2, respectively. The characterization of PNUTL1 and PNUTL2 represents a fundamental step in completing the characterization of the entire family of human septin genes.

A novel epithelial wound-related gene is abundantly expressed in developing rat cornea and skin

We previously used mRNA differential display and identified a novel gene that is up regulated in the healing corneal epithelium. To understand the potential in vivo role of this gene, termed T4a, we cloned the full-length T4a cDNA and investigated its temporal and spatial transcription expression in healing rat corneas, as well as in developing cornea and skin. The displayed T4a cDNA was used to identify clones from a rat cDNA library derived from healing corneal epithelia. The cDNA clones were sequenced and the sequence was analyzed with the Blast program. In situ hybridization was performed using digoxigenin-labeled riboprobes and cryostat sections from healing and developing cornea as well as skin. The T4a cDNA had 2538 bp with an open reading frame of 2178 bp, consistent with a conceptual translation product of 725 amino acid residues, a calculated molecular mass of 83.1 kD and theoretical pI of 6.93. Although T4a exhibited no sequence homology with known genes in the GenBank, it matched a large number of Expressed Sequence Tags (ESTs) from human, mouse and rat tissue cDNA libraries; more than half of the murine T4a ESTs were from embryonic DNA libraries. Sequence analysis revealed numerous phosphorylation and myristoylation sites in the deduced amino acid sequence of T4a. In the wounded rat cornea, intensive T4a mRNA staining was observed in the epithelium at all stages of re-epithelialization indicating that the expression of T4a is wound-related. In 7 day old mice, an abundant level of T4a transcripts was found in the epidermis and hair follicles, as well as in the corneal epithelial layer. High levels of T4a mRNA staining persisted in the developing postnatal corneal epithelia. In contrast, weak mRNA staining was only detected in the basal layer of the adult epidermis and corneal epithelium. These results indicate that T4a expression correlates with re-epithelialization of the cornea and maturation of the cornea and skin, suggesting a role for this gene in epithelial development, differentiation, and wound healing.

A novel epithelial wound-related gene is abundantly expressed in developing rat cornea and skin

Purpose. We previously used mRNA differential display and identified a novel gene that is up regulated in the healing corneal epithelium. To understand the potential in vivo role of this gene, termed T4a, we cloned the full-length T4a cDNA and investigated its temporal and spatial transcription expression in healing rat corneas, as well as in developing cornea and skin. Methods. The displayed T4a cDNA was used to identify clones from a rat cDNA library derived from healing corneal epithelia. The cDNA clones were sequenced and the sequence was analyzed with the Blast program. In situ hybridization was performed using digoxigenin-labeled riboprobes and cryostat sections from healing and developing cornea as well as skin. Results. The T4a cDNA had 2538 bp with an open reading frame of 2178 bp, consistent with a conceptual translation product of 725 amino acid residues, a calculated molecular mass of 83.1 kD and theoretical pI of 6.93. Although T4a exhibited no sequence homology with known genes in the GenBank, it matched a large number of Expressed Sequence Tags (ESTs) from human, mouse and rat tissue cDNA libraries; more than half of the murine T4a ESTs were from embryonic DNA libraries. Sequence analysis revealed numerous phosphorylation and myristoylation sites in the deduced amino acid sequence of T4a. In the wounded rat cornea, intensive T4a mRNA staining was observed in the epithelium at all stages of re-epithelialization indicating that the expression of T4a is wound-related. In 7 day old mice, an abundant level of T4a transcripts was found in the epidermis and hair follicles, as well as in the corneal epithelial layer. High levels of T4a mRNA staining persisted in the developing postnatal corneal epithelia. In contrast, weak mRNA staining was only detected in the basal layer of the adult epidermis and corneal epithelium. Conclusion. These results indicate that T4a expression correlates with re-epithelialization of the cornea and maturation of the cornea and skin, suggesting a role for this gene in epithelial development, differentiation, and wound healing.

8] Analysis of galactolipids and UDP-galactose: Ceramide galactosyltransferase

Publisher Summary Glycosphingolipids are enriched in the outer leaflet of the plasma membrane of most eukaryotic cells where they are thought to be involved in cell recognition and signaling. Although glycosphingolipids constitute only a few mol% of the lipids in most membranes, they are major components of the myelin sheath, where GalCer and SGalCer are involved in axonal insulation, myelin function, and stability. The apical plasma membrane of epithelial cells in the gastrointestinal and urinary tracts is enriched in glycosphingolipids. In rodents, these are typically glucolipidsy whereas in humans most are galactolipids. Glycosphingolipids play a structural role in rigidifying and protecting the apical cell surface. Their role in sorting lipids and proteins to various membranes along the exocytotic and endocytotic transport routes is not fully understood. The foremost enzyme involved in the biosynthesis of galactosphingolipids is the UDP-galactose:ceramide galactosyltransferase, CGalT or GalT-1. CGalT catalyzes the transfer of galactose from UDP-galactose to Cer yielding GalCer and has relatively promiscuous substrate specificity. Whether there are one or more CGalT enzymes with distinct specificity and cellular localization has been a controversial issue. The localization of CGalT has long been enigmatic. The enzyme is exclusively localized to the endoplasmic reticulum (ER) by immunogold electron microscopy on ultrathin cryosections. CGalT is a high mannose-type glycoprotein that is N-glycosylated at Asn-78 and Asn-333 and contains a putative carboxy terminal Lys-Lys-Val-Lys ER-retrieval signal. The conceptual translation product exhibits no amino acid sequence similarity with other glycosyltransferases. Instead, CGalT is related to the superfamily of UDP-glucuronosyltransferases.

Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti.

The gene encoding sialokinin I, the principal vasodilatory peptide of Aedes aegypti, has been isolated and characterized. Degenerate oligonucleotide primers based on peptide amino acid sequence were used to amplify a gene fragment from messenger RNA (mRNA) isolated from female salivary glands. The amplification product was used to probe a salivary gland complementary DNA (cDNA) library, and a number of corresponding cDNAs were isolated and their primary sequence determined. Analysis of the conceptual translation product of a 406-bp cDNA indicates that sialokinin I is expressed as a preprosialokinin and is subsequently post-translationally processed to the active peptide. Northern analysis revealed a 490-bp transcription product expressed exclusively in female salivary glands, and hybridization in situ of probes to RNA in whole tissues localized gene expression to the medial lobe of female salivary glands. Screening of an Ae. aegypti genomic library with the cDNA resulted in the isolation of a clone containing the gene, designated Sialokinin I (Sia I). Comparison of the cDNA with the genomic clone reveals two introns of 62 bp and 833 bp. Primer extension analysis showed that several transcription initiation sites are present. Southern analysis of genomic DNA shows that Sia I is most probably a single-copy gene. Similarities of the Sia I gene product with other genes are confined to the region encoding the active decapeptide.

Molecular Cloning of the Human Gene, PNKP, Encoding a Polynucleotide Kinase 3′-Phosphatase and Evidence for Its Role in Repair of DNA Strand Breaks Caused by Oxidative Damage

Mammalian polynucleotide kinases catalyze the 5'-phosphorylation of nucleic acids and can have associated 3'-phosphatase activity, predictive of an important function in DNA repair following ionizing radiation or oxidative damage. The sequences of three tryptic peptides from a bovine 60-kDa polypeptide that correlated with 5'-DNA kinase and 3'-phosphatase activities identified human and murine dbEST clones. The 57.1-kDa conceptual translation product of this gene, polynucleotide kinase 3'-phosphatase (PNKP), contained a putative ATP binding site and a potential 3'-phosphatase domain with similarity to L-2-haloacid dehalogenases. BLAST searches identified possible homologs in Caenorhabditis elegans, Schizosaccharomyces pombe, and Drosophila melanogaster. The gene was localized to chromosome 19q13.3-13.4. Northern analysis indicated a 2-kilobase mRNA in eight human tissues. A glutathione S-transferase-PNKP fusion protein displayed 5'-DNA kinase and 3'-phosphatase activities. PNKP is the first gene for a DNA-specific kinase from any organism. PNKP expression partially rescued the sensitivity to oxidative damaging agents of the Escherichia coli DNA repair-deficient xth nfo double mutant. PNKP gene function restored termini suitable for DNA polymerase, consistent with in vivo removal of 3'-phosphate groups, facilitating DNA repair.

Cloning and characterization of the rat and human phosducin-like protein genes: structure, expression and chromosomal localization

We isolated and characterized the rat gene encoding phosducin-like protein (PhLP), a putative heterotrimeric G protein modulator. The transcription start site was mapped by primer extension. The putative promoter region lacked a TATA sequence but contained a potential initiator element. Two splice variants were identified by RT-PCR of rat brain RNA, potentially generating either the full length or an amino-truncated protein. Only the full-length protein was immunodetected in all mouse tissues surveyed. Comparison of the conceptual translation product of the rat PhLP gene with those from human and Drosophila clones shows a striking conservation in the amino-terminal region of PhLP from these species. This contrasts with the relatively low degree of homology between PhLP and phosducin in this region, suggesting a functional role for this portion of the PhLP protein. Finally, we mapped the human PhLP gene by PCR analysis of somatic cell hybrids and the Stanford G3 radiation hybrid panel. The human PhLP gene (PDCL) is located on chromosome 9, linked to the polymorphic markers D9S1876 and D9S1674 (66–71 cM).

A conserved His-Asp signal response regulator-like gene in Heterosigma akashiwo chloroplasts.

Regulation of gene expression in plastids may involve molecular components conserved from cyanobacteria-like ancestors. Among prokaryotes, genes are commonly regulated at the transcriptional level by 'two-component' or 'His-Asp' signal transducers, consisting of a 'sensor kinase', which autophosphorylates at a conserved histidine residue, and a cognate response regulator, which is phosphorylated by the sensor kinase at a conserved aspartate residue. A putative His-Asp response regulator gene (trg1: transcriptional regulatory gene 1) has been identified in the estuarine raphidophytic alga Heterosigma akashiwo. The chloroplast-encoded trg1 is 693 bp in length, contains no introns, and yields a conceptual translation product of 231 amino acids, with a predicted mass of 27 kDa. Homology searches suggest that Heterosigma trgl has an omnpR-like identity within the DNA-binding His-Asp family of response regulators. trg1 contains both the phosphorylation and DNA-binding domains which are present in prokaryote response regulators. Quantitative competitive RT-PCR showed that Heterosigma trg1 is expressed at low levels (5 microg per g total RNA). In contrast, psbA (a photosystem II component) transcript is abundant (60 mg per g total RNA). Cell cycle analysis showed that psbA abundance oscillates in response to light but trg1 mRNA levels are invariant. We hypothesize that a His-Asp phosphorelay mechanism may affect chloroplast genome transcription in a manner similar to bacterial signal transduction pathways in which 'sensor kinase' and cognate 'response regulator' proteins interact.

A lysozyme in the salivary glands of the malaria vector Anopheles darlingi.

A cDNA encoding a lysozyme expressed specifically in the salivary glands of the malaria vector mosquito, Anopheles darlingi, was isolated by differential screening an adult female salivary gland library with abdomen and salivary gland cDNAs. The primary nucleic acid sequence of the cDNA contains a deduced coding region of 429 nucleotides and 5'- and 3'-end non-transcribed regions. A signal peptide of twenty-three amino acids and a mature protein of 120 amino acids are evident in the conceptual translation product. The results of RT-PCR experiments indicated that in adult mosquitoes this gene is expressed specifically in the salivary glands. Lysozyme enzymatic activity was detected in the salivary glands and abdomens of adult mosquitoes, but the pH optimum differed for each tissue and this was interpreted to indicate the presence of more than one enzyme, each being expressed in a different tissue. The salivary gland lysozyme may be involved in protection against bacterial infection in the anterior portion of the mosquito digestive tract.