Unique Amino Acid Sequence Research Articles

Duplication, balancing selection and trans-species evolution explain the high levels of polymorphism of the DQA MHC class II gene in voles (Arvicolinae)

Major histocompatibility complex (MHC) genes play important role in host-parasite interactions and parasites are crucial factors influencing the population dynamics of hosts. We described the structure and diversity of exon 2 of the MHC class II DQA gene in three species of voles (Arvicolinae) exhibiting regular multi-annual fluctuations of population density and analysed the processes leading to the observed MHC polymorphism. By using cloning-sequencing methodology and capillary electrophoresis-single strand conformation polymorphism, we described seven sequences in the water, eight in the common, and seven in the bank voles coming from an area of 70 km(2) around the Nozeroy canton in the Jura Mountains (Franche Comté, France). All exon 2 sequences translate to give unique amino acid sequences and positive selection was found to act very intensively on antigen binding sites. We documented the presence of recombination at vole DQA region but its importance in generating allelic polymorphism seems to be relatively limited. For the first time within rodents, we documented the duplication of the DQA gene in all three species with both copies being transcriptionally active. Phylogenetic analysis of allelic sequences revealed extensive trans-species polymorphism within the subfamily although no alleles were shared between species in our data set. We discuss possible role of parasites in forming the recent polymorphism pattern of the DQA locus in voles.

Protein Chemotaxonomy. XIII. Amino Acid Sequence of Ferredoxin from Panax ginseng

The complete amino acid sequence of [2Fe-2S] ferredoxin from Panax ginseng (Araliaceae) has been determined by automated Edman degradation of the entire S-carboxymethylcysteinyl protein and of the peptides obtained by enzymatic digestion. This ferredoxin has a unique amino acid sequence, which includes an insertion of Tyr at the 3rd position from the amino-terminus and a deletion of two amino acid residues at the carboxyl terminus. This ferredoxin had 18 differences in its amino acid sequence compared to that of Petroselinum sativum (Umbelliferae). In contrast, 23-33 differences were observed compared to other dicotyledonous plants. This suggests that Panax ginseng is related taxonomically to umbelliferous plants.

The facial motor nucleus transcriptional program in response to peripheral nerve injury identifies Hn1 as a regeneration‐associated gene

Facial nerve axotomy (FNA) is a well-established experimental model of motoneuron regeneration. After peripheral nerve axotomy, a sequence of events including glial activation and axonal regrowth leads to functional recovery of the afflicted pool of motoneurons. Using microarray analysis we identified an increase in the expression of 60 genes (at a false discovery rate of 0.1, genes were significant P < 0.004) within the facial nucleus as a consequence of nerve injury. In situ hybridization analysis validated the increased expression of many of these axotomy-induced genes. One specific gene, encoding a unique primary amino acid sequence, termed hemopoietic- and neurologic-expressed sequence-1 (Hn1), was evaluated more extensively using several additional nerve injury paradigms. Hn1 mRNA was upregulated in injured facial motoneurons in both rats and mice. Sustained upregulation of Hn1 mRNA was evident after nerve resection whereas levels of Hn1 mRNA returned to baseline in animals subjected to nerve crush or nerve transection. Hn1 was also increased in the dorsal motor nucleus and the nucleus ambiguous after vagus nerve axotomy, another regeneration model. No upregulation of Hn1 expression was observed, however, in two nonregeneration models: FNA in newborn rats and rubrospinal tractotomy. Hn1 mRNA was ubiquitous in the developing central nervous system whereas its expression in adult brain was confined to neurons of the hippocampus, cortex and cerebellum. These findings identify Hn1 as a gene associated with nervous system development and nerve regeneration.

Identification of Four Novel Exon 5 Splice Variants of the Mouse μ-Opioid Receptor Gene: Functional Consequences of C-Terminal Splicing

The rat mu-opioid receptor clone in which novel exon 5 was found in the place of exon 4 (MOR-1B) was one of the first MOR-1 variants described. We now have identified the mouse homolog of the rat MOR-1B as well as four additional variants derived from splicing from exon 3 into different sites within exon 5. The sequences of all of the variants were identical except for the intracellular tip of the C terminus encoded by exon 5, where each variant predicted a unique amino acid sequence ranging from 2 to 39 amino acids. All of the mMOR-1B variants were selective for mu-opioids in receptor-binding assays, as anticipated, because they all have identical binding pockets defined by the transmembrane domains. However, the relative potency and efficacy of mu-agonists to each other varied from variant to variant in guanosine 5'-O-(3-[35S]thio)triphosphate-binding studies, as shown by morphine-6beta-glucuronide, which was the most efficacious agent against mouse MOR-1B1 (mMOR-1B1) and the least efficacious agent against mMOR-1B2. mMOR-1B4 was quite unusual. Although mMOR-1B4 was mu-selective in receptor-binding studies and antagonists labeled mMOR-1B4 well, the binding affinities of most of the mu-agonists were far lower than those seen with mMOR-1, suggesting that the 39 amino acids at the C terminus of mMOR-1B4 influences the conformation of the receptor and its ligand recognition site itself either directly or through its interactions with other proteins. In conclusion, alterations in the amino acid sequence of the C terminus do not alter the mu-specificity of the receptor but they can influence the binding characteristics, efficacy, and potency of mu-opioids.

Identification of novel targeting peptides for human ovarian cancer cells using “one-bead one-compound” combinatorial libraries

Using "one-bead one-compound" combinatorial chemistry technology, we generated random peptide libraries containing millions of 90 mum TentaGel beads, each with its own unique amino acid sequence. A cyclic random 8-mer library was screened with CAOV-3 (a human ovarian adenocarcinoma cell line) and beads with a unique ligand that bind to the cell surface receptors were coated by one or more layers of cells. These positive beads were isolated, stripped, and microsequenced. Several peptide motifs were identified from these screenings, some of which were novel and unique, e.g., cDGX(4)GX(6)X(7)c. Structure-activity relationship studies of this peptide revealed that the l-aspartate residue at position 2, the two glycines at positions 3 and 5, and the two d-cysteines at the amino and COOH terminus are critical for activity. In addition, a hydrophobic residue was preferred at position X(4), whereas amino acids at positions X(6) and X(7) were more variable. Binding of this peptide to a number of different cancer cell lines and normal cells was also determined and we observed that peptides with this motif bound preferentially to three other human ovarian cancer cell lines (ES-2, SKOV-3, and OVCAR-3) as well as a human glioblastoma cancer cell line (A172). Structural analysis of the peptides using high-resolution nuclear magnetic resonance spectroscopy revealed strong conformational similarity among all peptides with cX(1)GX(4)GX(6)X(7)c motif. Blocking study with a panel of anti-integrin antibodies strongly suggests alpha3 integrin present on these ovarian adenocarcinoma cells is the target receptor for this peptide.

Cloning and expression of hypoxia-inducible factor 1α from the hibernating ground squirrel, Spermophilus tridecemlineatus

Mammalian hibernation is associated with apnoic breathing patterns and a hypoxia–hypothermia connection has been suggested as part of the mechanism by which body temperature is reduced as animals enter torpor. Hence, we hypothesized that changes in the expression of the hypoxia inducible factor (HIF-1) may potentially be involved in regulating hibernation-responsive gene targets. The expression of the alpha subunit of HIF-1 was quantified at both gene and protein levels in four organs of the thirteen-lined ground squirrel, Spermophilus tridecemlineatus. Reverse transcription-PCR showed no change in hif-1α transcript levels in the liver, lung, skeletal muscle or brown adipose tissue of euthermic versus hibernating animals but HIF-1α protein levels were elevated by 60–70% in the two organs responsive for thermogenesis (brown adipose and skeletal muscle). Furthermore, assessment of DNA binding by HIF-1 in nuclear extracts from brown adipose revealed 6-fold higher levels in hibernator tissue than in euthermic controls suggesting increased expression of HIF-1 responsive genes during hibernation. The complete nucleotide sequence of hif-1α from ground squirrels, the first hif-1α sequence amplified from a hibernating mammal, was obtained using PCR amplification and 3′ and 5′ RACE. Amino acid sequence analysis revealed 90–95% identity with the HIF-1α protein from other mammals. Several unique amino acid sequence substitutions were identified that may affect protein conformation and could possibly function to counteract low temperature effects on HIF-1α conformation at near 0 °C body temperatures during torpor.

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.

The favorable features of tryptophan synthase for proving Beadle and Tatum's one gene-one enzyme hypothesis.

LAST year we celebrated the 100th anniversary of the birth of George Beadle. He, with Edward Tatum, in 1941, proposed one of the major conceptual advances in biology in the 20th century: the existence of a one gene–one enzyme relationship (Beadle and Tatum 1941).2 Unfortunately, like many biologists proposing exciting hypotheses, they could not provide the experimental evidence that would prove they were correct. At that time, the existing knowledge about gene and enzyme structure was inadequate to permit the relationship's verification. George Beadle was aware of this and undoubtedly regretted his inability to provide support for their hypothesis. In a retrospective article written for Phage and the Origins of Molecular Biology, Beadle (1966)(p. 30) states that at the Cold Spring Harbor Symposium of 1951 “the number whose faith in one gene–one enzyme remained steadfast could be counted on the fingers of one hand—with a couple of fingers left over.” Why? Why was their proposed basic relationship not accepted? Why could they not provide proof for their hypothesis? Beadle and Tatum's principal difficulty was that in the 1940s we knew relatively little about the chemical nature of genetic material. The prevailing view then was that genetic material might be protein. Existing information on DNA structure suggested that it consisted of repetitive nucleotide sequences. If correct, it would be unlikely that DNA could specify the many different proteins present in each organism. Also, it was not yet firmly established that polypeptides consist of linear sequences of amino acids. Therefore, during the early years following Beadle and Tatum's proposal of the one gene–one enzyme relationship, our understanding of the structural features of genes and enzymes was insufficient to allow formulation of a finite molecular relationship. Advances in the 1950s dealt with most of these issues and suggested a plausible experimental strategy that could be applied in comparing the structural features of a gene with that of its corresponding protein. One of the earliest relevant findings, by Avery et al. (1944), suggested that the pneumococcal transforming principle—presumably the genetic material of the organism—was very likely DNA (see Lederberg 1994). Unfortunately, the significance of this finding was not appreciated initially; questions were raised about the purity of the transforming principle, our understanding of the transformation process was incomplete, and doubts existed that pneumococcal genetic material would be structurally comparable to the genetic material of higher organisms. Somewhat later, in the early 1950s, Al Hershey and Martha Chase presented their isotopic labeling studies with bacteriophage-infected Escherichia coli. Their results provided what was considered convincing evidence that phage genetic material is in fact DNA (Hershey and Chase 1952; see Stahl 1998). Most importantly, in 1953, Jim Watson and Francis Crick described the double-helical structure of DNA (Watson and Crick 1953). Their contribution changed forever how genetic material would be viewed by everyone. The realization that each gene probably consists of a linear sequence of nucleotides prompted subsequent thought on the existence of a genetic code, an essential consideration with a direct bearing on the gene-enzyme relationship. During this period Matt Meselson and Frank Stahl established that DNA is replicated by synthesis of complementary DNA strands, revealing the elegant features of DNA structure that provide for its exact replication (Meselson and Stahl 1958). Also of great importance, Fred Sanger showed that an insulin polypeptide consists of a unique linear sequence of amino acids (Sanger 1952; Stretton 2002). The significance of the contributions of Sanger, Erwin Chargaff, and others are described in an article by Horace Judson (Judson 1993). The discoveries cited, and observations by others supporting their conclusions, redefined the gene–enzyme relationship of Beadle and Tatum as the gene-protein colinearity hypothesis. Despite these advances in the 1950s, the technology that would have allowed either isolation of a gene or determination of its nucleotide sequence was lacking. Furthermore, since the concept of the genetic code was yet to be developed, knowledge of Watson and Crick's double-helical structure of DNA was initially of little help to those wishing to experimentally address the structural relationship between a gene and the enzyme it was proposed to specify. In the late 1950s Seymour Benzer demonstrated that one could construct a linear fine-structure genetic map by crossing mutants with genetically separable alterations in the same genetic region (Benzer 1957). His findings were consistent with the interpretation that a gene consists of a specific linear sequence of nucleotides. At approximately the same time, Vernon Ingram identified the amino acid change in the hemoglobin of humans with sickle cell anemia, and he developed the protein “fingerprinting” technique, a method that could be applied in detecting the single amino acid change in any mutant protein (Ingram 1958; see Ingram 2004). These two advances provided an excellent pair of experimental approaches that could be applied immediately in testing the gene–protein colinearity concept.

Search for peptide sequences involved in human antisperm antibody-mediated male immunoinfertility by using phage display technology

The aim of the present study was to delineate peptide sequences against which antisperm antibodies (ASA) are raised in immunoinfertile men. Using the phage display technology, seven unique and novel dodecamer amino acid sequences were identified that reacted with the sera of immunoinfertile men. The peptides were synthesized based upon these amino acid sequences and examined for their immunoreactivity with sera from ASA-positive immunofertile men (n = 15) and ASA-negative fertile men (n = 18) for IgM, IgG, and IgA class of antibody in the enzyme-linked immunosorbent assay (ELISA). All the seven synthetic peptides showed a significantly (P < 0.001) higher mean absorbance values for IgG and/or IgA class of antibody with the immunoinfertile sera compared to fertile control sera. Three of the seven peptides demonstrated a stronger reaction (>2 SD units) with 27%-40% of immunoinfertile sera compared to fertile controls. These peptide sequences may find applications in the specific diagnosis and treatment of immunoinfertility and in contraceptive vaccine development. The phage display technique provides an exciting and novel technology to delineate sperm epitopes involved in immunoinfertility.

High level expression and single-step purification of hexahistidine-tagged l-2-hydroxyisocaproate dehydrogenase making use of a versatile expression vector set

Affinity tags as fusions to the N- or C-terminal part of proteins are valuable tools to facilitate the production and purification of proteins. In many cases, there may be the necessity to remove the tag after protein preparation to regain activity. Removal of the tag is accomplished by insertion of a unique amino acid sequence that is recognized and cleaved by a site specific protease. Here, we report the construction of an expression vector set that combines N- or C-terminal fusion to either a hexahistidine tag or Streptag with the possibility of tag removal by factor Xa or recombinant tobacco etch virus protease (rTEV), respectively. The vector set offers the option to produce different variants of the protein of interest by cloning the corresponding gene into four different Escherichia coli expression vectors. Either immobilized metal affinity chromatography or streptactin affinity chromatography can be used for the one-step purification. Furthermore, we show the successful application of the expression vector for C-terminal hexahistidine tagging. The expression and purification of His-tagged l-2-hydroxyisocaproate dehydrogenase yields fully active enzyme. The tag removal is here accomplished by a derivative of rTEV.

Regulation of Bin1 SH3 domain binding by phosphoinositides.

Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P(2) in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5)P(2)-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein-protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.

Cloning and characterization of an mRNA encoding a novel G protein α-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G sα (pfG sα) from the pearl oyster was isolated. The G sα cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus ( Octopus vulgaris) G sα. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG sα has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G sα mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG sα in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG sα that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G sα in MC3T3-E1 cells. These results indicated that G sα may be involved in regulation of physiological function, particularly in biological biomineralization.

Post-proteomic identification of a novel phage-encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes.

The M1T1 strain remains the most frequently isolated strain from group A streptococcal (GAS) infection cases worldwide. We previously reported that M1T1 differs from the fully sequenced M1 SF370 strain. To better understand the reason for the persistence and increased virulence of M1T1, we analysed its secreted proteome and identified two virulence proteins that are not present in the sequenced M1 SF370 strain: streptococcal pyrogenic exotoxin A (SpeA) and a streptodornase D (SdaD) homologue. In the present study, we determined the nucleotide sequence of the M1T1 streptodornase and found that its deduced amino acid sequence is highly similar to other streptococcal streptodornases, and is most closely related to the SdaD of GAS strain M49. M1T1 Sda shares two highly conserved domains with several DNases and putative DNases in streptococci; however, it possesses a unique C-terminal amino acid sequence. Thus, we named the protein Sda1, and we detected the presence of the sda1 gene in 16 M1T1 clinical isolates. The cloned and expressed Sda1 degrades both streptococcal and mammalian DNA at physiological pH. Amino acid similarity analyses of known GAS deoxyribonucleases suggest that Sda1 may be a chimeric protein created through recombination events. Moreover, a natural mutation that resulted in longer Sda1 and SdaD as compared to other GAS DNases was found to confer increased activity on the protein. Analysis of the sequences flanking sda1 determined that it is carried by a prophage or a prophage-like element inserted in the tRNA-Ser gene of M1T1 GAS. Ongoing studies in our laboratory aim to determine the contribution of Sda1 to the virulence of this globally disseminated M1T1 strain.

A new type of antimicrobial protein with multiple histidines from the hard tick, Amblyomma hebraeum.

A novel 11 kDa antimicrobial protein, named as hebraein, and having a unique amino acid sequence, was purified from the hemolymph of fed female Amblyomma hebraeum ticks. A full-length cDNA clone encoding hebraein was isolated from a cDNA library made from tick synganglia. Hebraein consists of 102 amino acids, including 6 cysteine residues; has 9 histidines in its C-terminal domain that are mainly present as HX repeats; and has no significant similarity to any known protein. The secondary structure prediction is very clearly all alpha-helical (4-6 helices) except for a very short extension at the C terminus. Such high alpha-helical content is quite different from known antimicrobial proteins. Recombinant hebraein and a mutant lacking the histidine residues in the C-terminal domain were constructed and expressed. Assayed at the slightly acidic pH equivalent of fed female tick hemolymph, the wild-type and the histidine-rich recombinant hebraein had stronger antimicrobial activities than the histidine-deficient mutant. The pH-dependent properties of histidine-rich antimicrobial proteins may allow the design of agents that would function selectively in specific pH environments. The results from protein profiling of hemolymph, analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry combined with ProteinChip technology and RT-PCR analysis suggested that this antimicrobial protein was up-regulated by blood feeding. Our findings describe a new type of antimicrobial protein with multiple cysteine and histidine residues, and with unique secondary structure.

RalA but not RalB enhances polarized delivery of membrane proteins to the basolateral surface of epithelial cells.

RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins.

GTP cyclohydrolase I: purification, characterization, and effects of inhibition on nitric oxide synthase in nocardia species.

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH4), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a Km for GTP of 6.5 micromoles. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56 degrees C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH4 inhibited enzyme activity by 25% at a concentration of 100 micromoles. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a Ki of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO2- plus NO3-. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH4.

Trypsin-modulating oostatic factor: a potential new larvicide for mosquito control

Trypsin-modulating oostatic factor (TMOF), a mosquito decapeptide, terminates trypsin biosynthesis in the mosquito gut. The hormone is secreted from the ovary, starting 18 h after the blood meal, circulates in the hemolymph, binds to a gut receptor and stops trypsin biosynthesis by exerting a translational control on trypsin mRNA. Because of the unique primary amino acid sequence of the hormone (YDPAPPPPPP) and its stable three-dimensional conformation, TMOF is not degraded by gut proteolytic enzymes and can traverse the gut epithelial cells into the hemolymph of adults and larvae. Using this unique property, hormone fed to different species of mosquito larvae stops food digestion and causes larval mortality. To determine the shortest amino acid sequence that can bind to the gut receptor and still cause high larval mortality, 25 analogues of TMOF were synthesized and tested. The tetrapeptide (YDPA) was as effective as the decapeptide, indicating that the binding to the gut receptor is at the N-terminus of the molecule. Cloning and expressing the hormone on the coat protein of tobacco mosaic virus (TMV) in Chlorella sp. and Saccharomyces cerevisiae cells and feeding the recombinant cells to mosquito larvae caused larval mortality. These results indicate that TMOF can be used as a new biorational insecticide against mosquito larvae.

Functional analysis of MOR-1 splice variants of the mouse mu opioid receptor gene Oprm.

A series of mu opioid receptor gene Oprm splice variants have been reported that differ only at their C-terminus. These variants all contain exons 1, 2, and 3 of the gene, the exons responsible for coding all seven transmembrane domains. Whereas MOR-1 also has exon 4 that encodes for an additional 12 amino acids at the tip of the C-terminus, the other MOR-1 variants have unique amino acid sequences distinct from those in MOR-1 due to alternative splicing. All these variants are mu-selective in binding assays. The current study explored the ability of these variants to stimulate [35S]GTPgammaS binding to assess them functionally. Only mu opioids stimulated [35S]GTPgammaS binding. Among the mu opioids we noted marked differences in their maximal stimulation among the clones. This was most prominent with beta-endorphin, which stimulated [35S]GTPgammaS binding in the MOR-1E expressing cells to a greater degree than [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO; 130%) and was far less effective than DAMGO in MOR-1C cells (44%). The rank order of maximal stimulation of the drugs varied among the clones as well. Dynorphin A, beta-endorphin and morphine were most effective in stimulating [35S]GTPgammaS binding in MOR-1E, while M6G and fentanyl were most effective in MOR-1 expressing cells. The potency (EC50) of some of the drugs also varied extensively among the clones, with a poor correlation between the potency of the drugs to stimulate [35S]GTPgammaS binding and their binding affinity. Together, these findings reveal marked functional differences among the variants that only can be explained by their structural differences at the tip of their C-terminus.

Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase epsilon (Pol epsilon) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol epsilon synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of </=2 x 10-5 and </=5 x 10-7, respectively. Pol epsilon lacking 3' --> 5' exonuclease activity is less accurate to a degree suggesting that wild type Pol epsilon proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol epsilon is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T.dTTP, T.dCTP, and C.dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol epsilon differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol epsilon in vitro and substitutions occurring in vivo in a yeast strain defective in Pol epsilon proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol epsilon polymerase activity in DNA replication.

The Fifth Carbohydrate Bioengineering Meeting was held in Groningen, The Netherlands, April 6–9, 2003

An alpha-galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first alpha-galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other alpha-galactosidases. Highest homology was found with alpha-amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant alpha-galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl-alpha-galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable alpha-galactosidase with a temperature optimum of 115 degrees C and a half-life time of 15 hours at 100 degrees C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by the addition of azide or formate. It is concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified.