Potential Transmembrane Helices Research Articles

Verticillium dahliae VdPBP1 Transcription Factor Is Required for Hyphal Growth, Virulence, and Microsclerotia Formation.

Verticillium dahliae, a fungal pathogen that affects more than 200 plant species, including tomatoes, requires specific proteins for its early steps in plant infection. One such crucial protein, VdPBP1, exhibits high expression in the presence of tomato roots. Its 313-amino acid C-terminal section restores adhesion in nonadhesive Saccharomyces cerevisiae strains. To uncover its role, we employed a combination of bioinformatics, genetics, and morphological analyses. Our findings underscore the importance of VdPBP1 in fungal growth and pathogenesis. Bioinformatic analysis revealed that the VdPBP1 gene consists of four exons and three introns, encoding a 952-codon reading frame. The protein features a 9aaTAD domain, LsmAD, and PAB1 DNA-binding sites, as well as potential nuclear localization and transmembrane helix signals. Notably, the deletion of a 1.1 kb fragment at the gene's third end impedes microsclerotia formation and reduces pathogenicity. Mutants exhibit reduced growth and slower aerial mycelial development compared to the wild type. The VdPBP1 deletion strain does not induce disease symptoms in tomato plants. Furthermore, VdPBP1 deletion correlates with downregulated microsclerotia formation-related genes, and promoter analysis reveals regulatory elements, including sites for Rfx1, Mig1, and Ste12 proteins. Understanding the regulation and target genes of VdPBP1 holds promise for managing Verticillium wilt disease and related fungal pathogens.

A novel B- and helper T-cell epitopes-based prophylactic vaccine against Echinococcus granulosus

Introduction: In this study, we targeted the worm stage of Echinococcus granulosus to design a novel multi-epitope B- and helper T-cell based vaccine construct for immunization of dogs against this multi-host parasite. Methods: The vaccine was designed based on the local Eg14-3-3 antigen (Ag). DNA samples were extracted from the protoscoleces of the infected sheep’s liver, and then subjected to the polymerase chain reaction (PCR) with 14-3-3 specific forward and reverse primers. For the vaccine designing, several in silico steps were undertaken. Three-dimensional (3D) structure of the local Eg14-3-3 Ag was modeled by EasyModeller software. The protein modeling accuracy was then analyzed via various validation assays. Potential transmembrane helix, signal peptide, post-translational modifications and allergenicity of Eg14-3-3 were evaluated as the preliminary measures of B-cell epitopes (BEs ) prediction. Having used many web-servers, a well-designed process was carried out for improved prediction of BEs. High ranked linear and conformational BEs were utilized for engineering the final vaccine construct. Possible T-helper epitopes (TEs) were identified by the molecular docking between 13-mer fragments of the Eg14-3-3 Ag and two high frequent dog class II MHC alleles (i.e., DLA-DRB1*01101 and DRB1*01501). The epitopes coverage was evaluated by Shannon’s variability plot. Results: The final designed construct was analyzed based on different physicochemical properties, which was then codon optimized for high-level expression in Escherichia coli k12. This minigene construct is the first dog-specific epitopic vaccine construct that is established based on TEs with high-binding affinity to canine MHC alleles. Conclusion: This in silico study is the first part of a multi-antigenic vaccine designing work that represents as a novel dog-specific vaccine against E. granulosus. Here, we present key data on the step-by-step methodologies used for designing this de novo vaccine, which is under comprehensive in vivo investigations.

Heterologous Production of Functional Chloroform Reductive Dehalogenase

BackgroundReductive dehalogenases (RDase) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). RDases are membrane‐associated proteins whose activity is dependent on protein‐associated cobalamin and iron‐sulfur clusters. The heterologous expression of respiratory RDases is reported to be extremely challenging. Dehalobacter strain UNSWDHB is an ORB capable of respiring chloroform (CF, or trichloromethane) as an electron acceptor for the generation of cellular energy and as a consequence, dechlorinates CF to dichloromethane. TmrA enzyme is responsible for this reaction (i.e. CF‐RDase) and has been purified from Dehalobacter strain UNSWDHB and partly characterized. This study explores heterologous expression of the same enzyme.MethodsWe cloned a codon optimized tmrA with an N‐terminal hexa‐His‐tag replacing the TAT signal sequence and potential transmembrane helix region into the pPT7 plasmid. The pPT7‐tmrA plasmid was transformed into Bacillus megaterium MS941 containing the pT7‐RNAP plasmid. Xylose‐inducible expression of tmrA was performed aerobically in TB media at 17°C. The cells were disrupted anaerobically using a French press. CF dechlorination activity of the soluble protein fraction was measured by gas chromatography using Ti(III)citrate and methyl viologen as electron donors.ResultsWe expressed the N‐His tagged TmrA protein in B. megaterium cells. Soluble protein fractions exhibited CF dechlorination activity in vitro. Further biochemical characterizations will be undertaken on the purified enzyme.ConclusionThis is the first report of heterologous expression of soluble and active respiratory RDase in B. megaterium. The CF reducing TmrA enzyme was expressed in cobalamin‐producing B. megaterium cells, and dichloromethane production could be demonstrated in the soluble protein fractions.Support or Funding InformationThe authors wish to acknowledge the University of New SouthWales (UNSW) for providing scholarship support for BJ.

Why is the biological hydrophobicity scale more accurate than earlier experimental hydrophobicity scales?

The recognition of transmembrane helices by the translocon is primarily guided by the average hydrophobicity of the potential transmembrane helix. However, the exact hydrophobicity of each amino acid can be identified in several different ways. The free energy of transfer for amino acid analogues between a hydrophobic media, for example, octanol and water can be measured or obtained from simulations, the hydrophobicity can also be estimated by statistical properties from known transmembrane segments and finally the contribution of each amino acid type for the probability of translocon recognition has recently been measured directly. Although these scales correlate quite well, there are clear differences between them and it is not well understood which scale represents neither the biology best nor what the differences are. Here, we try to provide some answers to this by studying the ability of different scales to recognize transmembrane helices and predict the topology of transmembrane proteins. From this analysis it is clear that the biological hydrophobicity scale as well scales created from statistical analysis of membrane helices perform better than earlier experimental scales that are mainly based on measurements of amino acid analogs and not directly on transmembrane helix recognition. Using these results we identified the properties of the scales that perform better than other scales. We find, for instance, that the better performing scales consider proline more hydrophilic. This shows that transmembrane recognition is not only governed by pure hydrophobicity but also by the helix preferences for amino acids, as proline is a strong helix breaker.

The mechanism of toxicity in HET-S/HET-s prion incompatibility.

Author SummaryFilamentous fungi have the potential for genetically distinct individuals to fuse, resulting in a cell with multiple nuclei known as a heterokaryon. This fusion event is controlled by genetic variants that determine the compatibility of the individuals, such that the fusion of incompatible genotypes triggers a cell death reaction in the heterokaryon. We have investigated the molecular mechanism of toxicity in the HET-S/HET-s incompatibility system in the fungus P. anserina. HET-s is an infectious yet non-toxic protein (prion) whose interaction with the almost identical protein HET-S has been shown to re-localize HET-S to the cell periphery, an event that is associated with the death of heterokaryons that simultaneously contain both proteins. We find that the HET-s prion converts soluble HET-S into a protein that binds to and destabilizes lipid membranes. Furthermore, we identify a potential transmembrane helix that is normally buried within the soluble fold of HET-S and show that its presence is associated with toxicity. We conclude that upon interaction with a HET-s prion, the HET-S globular domain partially unfolds, exposing a previously buried transmembrane segment that targets HET-S to the membrane. Once there, it further oligomerizes into a structure that causes a loss of membrane integrity, reminiscent of the mode of action of pore-forming toxins.

PalI domain proteins of Saccharomyces cerevisiae and Candida albicans

The Rim9/PalI groups of proteins are members of the Sur7 family, all of which contain a signal sequence and a block of three potential trans-membrane helices. Multi-protein sequence comparisons among fungi suggest that there are two classes of Rim9/PalI proteins; longer proteins like PalI that contain a Sur7 domain and a C-terminal extension, and shorter proteins like Rim9 that contain essentially only the Sur7 domain. We have examined possible roles of the longer, PalI-like proteins of both Saccharomyces cerevisiae (Yol019w) and Candida albicans (Orf19.1510/Srd1), two species that also contain short Rim9 proteins required for alkaline-associated stress responses. Deletions of the long form genes did not create any significant stress response phenotype in either S. cerevisiae or C. albicans, nor did the deletions enhance any of the rim9 deletion effects when combined in a double mutant. Furthermore, challenges in C. albicans show RIM9 but not SRD1 is important for proper response and hyphal formation. It appears that in fungal species such as Aspergillus nidulans containing only a long-form PalI-like protein, this element functions in the process of stress response, while in fungi with both versions the response to stress function is limited to the short-form protein.

Identification of a polymorphic collagen-like protein in the crustacean bacteria Pasteuria ramosa

Pasteuria ramosa is a spore-forming bacterium that infects Daphnia species. Previous results demonstrated a high specificity of host clone/parasite genotype interactions. Surface proteins of bacteria often play an important role in attachment to host cells prior to infection. We analyzed surface proteins of P. ramosa spores by two-dimensional gel electrophoresis. For the first time, we prove that two isolates selected for their differences in infectivity reveal few but clear-cut differences in protein patterns. Using internal sequencing and LC/MS/MS, we identified a collagen-like protein named Pcl1a ( Pasteuria collagen-like protein 1a). This protein, reconstructed with the help of Pasteuria genome sequences, contains three domains: a 75-amino-acid amino-terminal domain with a potential transmembrane helix domain, a central collagen-like region (CLR) containing Gly-Xaa-Yaa (GXY) repeats, and a 7-amino-acid carboxy-terminal domain. The CLR region is polymorphic among the two isolates with amino-acid substitutions and a variable number of GXY triplets. Collagen-like proteins are rare in prokaryotes, although they have been described in several pathogenic bacteria, including Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis, closely related to Pasteuria species, in which they could be involved in the adherence of bacteria to host cells.

Identification and tissue-specific expression of a promethin-like homolog in amphioxus Branchiostoma belcheri

Promethins have been shown to be present in the vertebrates examined so far, yet little is known to date about them in invertebrates. Here we isolated a cDNA encoding a promethin-like homolog from the gut cDNA library of the amphioxus Branchiostoma belcheri, a cephalochordate occupying a nodal position transient from invertebrates to vertebrates. It contained a 504 bp open reading frame corresponding to a protein of 167 amino acids. Primary structural examination showed that the deduced promethin-like homolog was a transmembrane protein with three potential transmembrane helices, resembling the vertebrate promethins. Phylogenetic analysis showed that B. belcheri promethin-like homolog was located at the base of the vertebrate counterparts, suggesting that it represents the archetype of vertebrate promethins. Both Northern blotting and in situ hybridization histochemistry revealed a tissue-specific expression pattern of promethin-like gene, like that of mammalian promethins. This is the first report on invertebrate promethin-like homolog, paving the way for further insights into the evolution and function of promethins.

Analyses of the complete sequence of the genome of wheat Eqlid mosaic virus, a novel species in the genus Tritimovirus

The complete nucleotide sequence of the genome of wheat Eqlid mosaic virus (WEqMV) (excluding the poly A tail) comprised 9636 nucleotides including 5' and 3' noncoding regions of 137 and 172 nt, respectively. It contained a single ORF coding for a polyprotein of 3,109 amino acid residues and had a deduced genome organization typical of members of the family Potyviridae and with proteinase cleavage sites very similar to those of the members of the genus Tritimovirus. Pairwise and multiple alignments and phylogenetic analysis showed that WEqMV is a distinct species in the genus Tritimovirus. WEqMV and Wheat streak mosaic virus (WSMV) shared the greatest nucleotide sequence identity in the NIb and HC-Pro cistrons (63.2% and 60.8%, respectively) and the lowest sequence identity in the P1 and CP cistrons (51.2% and 51.1%, respectively). Sequence identity for the complete genome of WEqMV and WSMV was 56.8% at the nucleotide level and 50.7% at the amino acid level. WEqMV had 57.2% nucleotide identity and 50.6% amino acid identity with Oat necrotic mottle virus and 52.5% nucleotide identity and 45.5% amino acid identity with Brome streak mosaic virus. The relationship of WEqMV with other members of the family Potyviridae was more distant. Structural analysis of WEqMV protein showed presence of potential transmembrane helices in 6k1, 6k2, and P3 proteins.

The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage ϕWMY: Cloning, sequencing, expression, and mutational analysis in Escherichia coli

From the genome library of Staphylococcus warneri M, the two successive cell-lysis genes ( holWMY and lytWMY) were cloned and characterized. The lytWMY gene encoded a protein (LysWMY), whose calculated molecular mass and p I were 54 kDa and 8.95, respectively. When overproduced in Escherichia coli, lysWMY directed a protein of 45 kDa (smaller than the predicted molecular mass), having N-terminal 13 residues identical with those predicted from DNA. Comparative analysis revealed that LysWMY significantly resembles the putative N-acetylmuramoyl- l-alanine amidases encoded by the staphylococcal phages ϕ11, 80 alpha, and Twort. Examination of modular organization of LysWMY identified three putative domains CHAP (for d-alanyl-glycyl endopeptidase), amidase ( l-muramoyl- l-alanine amidase), and SH3 (cell wall recognition). Gene knockout analysis revealed that each of the two domains of CHAP and amidase was responsible for cell-lytic activity on a zymogram gel. Site-directed mutation of Cys29Ala, His92Ala, or Asn114Ala in the CHAP domain substantially reduced cell-lytic activity, suggesting that this Cys–His–Asn triad is crucial for the enzymatic function. On the other hand, the holWMY gene encoded a protein (HolWMY) with molecular mass and p I of 16 kDa and 4.36; this protein contained two potential transmembrane helices, resembling other predicted holins (a cytoplasmic membrane-disrupting protein) encoded by the S. aureus phage, ϕ11, 80 alpha, and Twort. Upon mitomycin C exposure of S. warneri M, a prophage (ϕWMY) was induced and the virion was examined under electron microscopy. PCR amplification and sequencing revealed the presence of the holWMY–lysWMY genes in the phage genome.

Molecular properties of the two-component cell lysis system encoded by prophage φgaY of Lactobacillus gasseri JCM 1131 T: cloning, sequencing, and expression in Escherichia coli

Shotgun cloning of the Lactobacillus gasseri JCM 1131 T whole DNA yielded two recombinant plasmids, p118gaY1 and p118gaY2, which directed cell lysis activity. Sequencing analysis revealed that the two plasmids carried almost identical inserted genes in following orders (truncated genes, in parentheses): in p118gaY1, ( orf149) -orf92-holgaY-lysgaY-orf35-attL-( mnaAgaY1); in p118gaY2, ( orfXgaY1) -orf169-orf149-orf92-holgaY-lysgaY-orf35-attP-( intgaY). The lysgaY-encoded protein (designated as LysgaY, 33.7 kDa) showed significant homology with putative muramidases (peptidoglycan-degrading enzyme) of the Lactobacillus phage φadh, Lj965, Lj928, LL-H, mv4, and mv1. By zymogram analysis, LysgaY overproduced in Escherichia coli exhibited lytic activity towards 17 Gram-positive bacterial strains, including lactobacilli, lactococci, and staphylococci. The holgaY-encoded protein (15.7 kDa) contained three potential transmembrane helices, resembling putative holins (cytoplasmic membrane-disrupting protein) of Lj928 and Lj965. On the other hand, another clone p118gaYR obtained by EcoRI-shotgun cloning carried the ( ptsCgaY1)- attR-( intgaY) genes. Three sequences, attL, attP, and attR, had a 47-bp common (core) sequence, and the core of attR was located in 3′ region of a potential tRNA Arg gene. These results suggested that (i) attL and attR are phage–host junctions, left- and right-arms, respectively, (ii) attP is a phage attachment site, and (iii) intgaY is an integrase gene for phage integration and/or excision. After mitomycin C-induction, phage particles were demonstrated by electron microscopy. The prophage (φgaY) is somewhat leaky in the host, and has the two-component lysis system (HolgaY–LysgaY), closely resembling that of Lj928 as well as Lj965.

Nucleotide sequence, genome organization and phylogenetic analysis of Strawberry pallidosis associated virus, a new member of the genus Crinivirus

The complete nucleotide sequence of Strawberry pallidosis associated virus (SPaV), a newly identified member of the genus Crinivirus, family Closteroviridae has been determined. RNA 1 is 8067 nucleotides long and encodes at least three open reading frames (ORFs). The first ORF (ORF 1a) specifies a multifunctional protein that has papain-like proteinase, methyltransferase and RNA helicase domains. The RNA-dependent-RNA polymerase is encoded in ORF 1b and is probably expressed by a +1 ribosomal frameshift. The 3' ORF of RNA 1 encodes a small protein with two potential transmembrane helices. RNA 2 is 7979 nucleotides long and encodes 8 ORFs, similar in amino acid sequence and arrangement with those of other criniviruses. SPaV encodes the largest structural protein of closteroviruses sequenced to date as the minor coat protein of the virus has molecular mass of approximately 80 kDa. The 3' non-translated regions share nucleotide sequence identities of about 56% and the predicted folding of the non-translated regions is similar. Phylogenetic analyses reveal that SPaV is related most closely to Abutilon yellows virus and Beet pseudo-yellows virus, another virus that has been identified recently to cause identical symptoms on strawberry indicator plants as SPaV.

Identification and analysis of gp116 and gp64 structural glycoproteins of yellow head nidovirus of Penaeus monodon shrimp.

Yellow head virus (YHV) is a major agent of disease in farmed penaeid shrimp. YHV virions purified from infected shrimp contain three major structural proteins of molecular mass 116 kDa (gp116), 64 kDa (gp64) and 20 kDa (p20). Two different staining methods indicated that the gp116 and gp64 proteins are glycosylated. Here we report the complete nucleotide sequence of ORF3, which encodes a polypeptide of 1666 amino acids with a calculated molecular mass of 185 713 Da (pI=6.68). Hydropathy analysis of the deduced ORF3 protein sequence identified six potential transmembrane helices and three ectodomains containing multiple sites for potential N-linked and O-linked glycosylation. N-terminal sequence analysis of mature gp116 and gp64 proteins indicated that each was derived from ORF3 by proteolytic cleavage of the polyprotein between residues Ala(228) and Thr(229), and Ala(1127) and Leu(1128), located at the C-terminal side of transmembrane helices 3 and 5, respectively. Comparison with the deduced ORF3 protein sequence of Australian gill-associated virus (GAV) indicated 83 % amino acid identity in gp64 and 71 % identity in gp116, which featured two significant sequence deletions near the N terminus. Database searches revealed no significant homology with other proteins. Recombinant gp64 expressed in E. coli with and without the C-terminal transmembrane region was shown to react with antibody raised against native gp64 purified from virions.

Definition of the Nuclear Encoded Protein Composition of Bovine Heart Mitochondrial Complex I: IDENTIFICATION OF TWO NEW SUBUNITS

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) from bovine heart is a complicated multisubunit, membrane-bound assembly. Seven subunits are encoded by mitochondrial DNA, and the sequences of 36 nuclear encoded subunits have been described. The subunits of complex I and two subcomplexes (Ialpha and Ibeta) were resolved on one- and two-dimensional gels and by reverse-phase high performance liquid chromatography. Mass spectrometric analysis revealed two previously unknown subunits in complex I, named B14.7 and ESSS, one in each subcomplex. Coding sequences for each protein were identified in data bases and were confirmed by cDNA cloning and sequencing. Subunit B14.7 has an acetylated N terminus, no presequence, and contains four potential transmembrane helices. It is homologous to subunit 21.3b from complex I in Neurospora crassa and is related to Tim17, Tim22, and Tim23, which are involved in protein translocation across the inner membrane. Subunit ESSS has a cleaved mitochondrial import sequence and one potential transmembrane helix. A total of 45 different subunits of bovine complex I have now been characterized.

Structure and developmental regulation of CONSTANS- LIKE genes isolated from Populus deltoides

Two CONSTANS- LIKE ( COL) genes, PdCO1 and PdCO2, were isolated from eastern cottonwood ( Populus deltoides Bartr. ex. Marsh. var. deltoides). PdCO1 is unique among all the characterized CONSTANS-LIKE genes in the plant kingdom in that the deduced protein sequence contains a putative signal peptide with a potential transmembrane helix at the N-terminal end. Current bioinformatics tools suggest that PdCO1 protein may be secreted. Immuno-blot analysis indicated that although the PdCO1 was abundant in leaf, it was not detected in the subtended bud. Temporal mRNA and protein expression of PdCO1 and PdCO2 was determined in the leaf and bud at the ninth node of shoots, where transition from vegetative to floral phase has been observed to occur. The expression of both genes in the leaf increased dramatically from March 30 to April 21 and decreased on May 9. The increased expression coincided with morphological changes occurring in the axillary meristem of the subtended bud and with increased abundance of PdCO1 protein in the leaf. These changes sequentially included (1) the cessation of leaf-primordium formation, (2) the transition of the axillary shoot apical meristem to an inflorescence meristem, (3) and the subsequent formation of bracts and floral meristems in the axils of bracts flanking the inflorescence meristem. The temporal expression of PdCO1 and PdCO2 prior to flower bud development suggests that these genes might be involved in floral induction.

Retention of the Yeast Sac1p Phosphatase in the Endoplasmic Reticulum Causes Distinct Changes in Cellular Phosphoinositide Levels and Stimulates Microsomal ATP Transport

The yeast phosphoinositide phosphatase Sac1p localizes to endoplasmic reticulum (ER) and Golgi membranes and has compartment-specific functions in these organelles. In this study we analyzed in detail the topology of Sac1p. Our data show that Sac1p is a type II transmembrane protein with a large N-terminal cytosolic domain, which is anchored in the membrane by the two potential transmembrane helices near the C terminus. Based on this topology, we created a mutation that caused retention of Sac1p in the ER and as a consequence showed specific alterations in cellular phosphoinositide levels. Our results suggest that Sac1p controls a pool of phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate in the ER. Retention of Sac1p in the ER also stimulates ATP transport into the ER lumen but causes the same Golgi-specific defects that are seen in a sac1 null mutant. Taken together this study provides evidence that Sac1p is an important 4-phosphatase in the ER controlling different aspects of ER-based protein processing and secretion.

Characterization of the murine gammaherpesvirus 68 ORF74 product: a novel oncogenic G protein-coupled receptor

Murine gammaherpesvirus (MHV-68) is well established as a small animal model for the study of gammaherpesviruses. The MHV-68 genome contains an open reading frame (ORF74) that has significant sequence homology with mammalian G-protein coupled receptors (GPCRs) and the GPCR from the related Kaposi's sarcoma-associated herpesvirus (KSHV). Here we show that the MHV-68 ORF74 is predicted to encode a GPCR since it has seven potential transmembrane helices and that it has other sequence motifs in common with GPCRS: Of interest is the observation that the sequence around a conserved arginine at the start of the second intracellular loop suggests that the ORF74 product may signal constitutively (agonist independent). Given that the ORF74 product is predicted to encode a GPCR we named it MHV-GPCR. In studies on the transcription of the MHV-GPCR, we determined that it was encoded on multiple early transcripts of 3.4, 4.4, 6.6 and 8.7 kb in size. At least one of these transcripts was bicistronic, containing the ORF encoding the Bcl-2 homologue also. In vivo, we found that MHV GPCR was expressed during acute infection but also during persistence, particularly in the lungs of infected mice. Immunofluorescence studies indicated that the MHV GPCR protein was expressed on the surface of cells in patches. Finally, like the KSHV GPCR, expression of the MHV GPCR resulted in transformation of NIH 3T3 cells. We surmise, therefore, that the MHV GPCR may act in concert with genes with which it is expressed such as vBcl-2 to enhance the growth and survival of MHV-68-infected cells.

Genomic organization and chromosomal localization of the murine 2 P domain potassium channel gene Kcnk8: conservation of gene structure in 2 P domain potassium channels

A 2 P domain potassium channel expressed in eye, lung, and stomach, Kcnk8, has recently been identified. To initiate further biochemical and genetic studies of this channel, we assembled the murine Kcnk8 cDNA sequence, characterized the genomic structure of the Kcnk8 gene, determined its chromosomal localization, and analyzed its activity in a Xenopus laevis oocyte expression system. The composite cDNA has an open reading frame of 1029 bp and encodes a protein of 343 amino acids with a predicted molecular mass of 36 kDa. Structure analyses predict 2 P domains and four potential transmembrane helices with a potential single EF-hand motif and four potential SH3-binding motifs in the COOH-terminus. Cloning of the Kcnk8 chromosomal gene revealed that it is composed of three exons distributed over 4 kb of genomic DNA. Genome database searching revealed that one of the intron/exon boundaries identified in Kcnk8 is present in other mammalian 2 P domain potassium channels genes and many C. elegans 2P domain potassium channel genes, revealing evolutionary conservation of gene structure. Using fluorescence in situ hybridization, the murine Kcnk8 gene was mapped to chromosome 19, 2B, the locus of the murine dancer phenotype, and syntenic to 11q11-11q13, the location of the human homologue. No significant currents were generated in a Xenopus laevis oocyte expression system using the composite Kcnk8 cDNA sequence, suggesting, like many potassium channels, additional channel subunits, modulator substances, or cellular chaperones are required for channel function.

Sp22D: a multidomain serine protease with a putative role in insect immunity

Serine proteases play critical roles in a variety of insect immune responses; however, few of the genes that code for these enzymes have been cloned. Here, we describe the molecular characterization of a serine protease gene from the mosquito Anopheles gambiae. Sp22D codes for a 1322 amino acid polypeptide with a complex domain organization. In addition to the carboxy terminal serine protease catalytic domain, Sp22D contains two putative chitin binding domains, a mucin-like domain, two low density lipoprotein receptor class A domains, and two scavenger receptor cysteine rich domains. A typical signal peptide sequence and a lack of potential transmembrane helices suggest that Sp22D is secreted. Sp22D is expressed constitutively in three immune-related cell types: adult hemocytes, fat body cells, and midgut epithelial cells. Wounding induces no changes in transcript abundance, but within 1 h after injection of bacteria, Sp22D mRNA increases 1.5-fold. Based on domain organization, tissue distribution, and transcriptional up-regulation in response to immune challenge, we suggest that Sp22D has an immune function. In addition, we predict that Sp22D is secreted into the hemolymph where it may interact with pathogen surfaces and initiate an immune response.

Cloning and expression of the genes involved in the production of and immunity against the bacteriocin lacticin RM

The production of lacticin RM, a novel bacteriocin produced by Lactococcus lactis subsp. lactis EZ26, is associated with the presence of a 6-kb plasmid, pHU1. The information necessary for lacticin RM production and immunity was localized to a 2.5-kb SalI– Eco47III fragment. Sequencing analysis of this fragment revealed the presence of six open reading frames (ORFs). Deletion and mutation analyses showed that orfX and orfY are not required for lacticin RM production or immunity, whereas the other ORFs ( lacA, lacF, lacG and lacI) are necessary for the bacteriocin’s production. Transcription analysis indicated that lacA, lacF and lacG are organized in an operon. lacA is probably the lacticin RM structural gene. It putatively encodes a 134-amino acid peptide, and it does not share homology with known bacteriocins. The deduced LacG protein is hydrophobic and consists of six potential trans-membrane helices. lacF encodes a conserved ATP-binding domain homologous to ABC transporters known in bacteriocin immunity systems. LacF and LacG may form an active ABC transporter. Gene-disruption mutations indicated that both are required for immunity against lacticin RM. lacI encodes a small cationic protein, which is required for the production of and immunity to lacticin RM. Protection was obtained only when lacF, lacG and lacI were present together.