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Functional and antigenic domains of the matrix (M1) protein of influenza A virus.

The membrane- and ribonucleocapsid (RNP)-binding domains of the matrix (M1) protein of influenza A virus (WSN strain) were partially mapped and characterized by reactivity with monoclonal antibodies (MAb) as well as by proteolytic cleavages and amino acid sequencing of the resulting peptides. Of two peptides formed by formic acid hydrolysis, a 9-kilodalton fragment at the amino-terminal third of the M1 protein was recognized by MAb M2-1C6 (to epitope 1), and a 15-kilodalton fragment at the carboxy-terminal two-thirds was recognized by MAb 289/4 (to epitope 2). Partial cleavage by staphylococcal V8 protease gave rise to a 16-kilodalton peptide, mapping to amino acid 8, which was recognized by MAbs to all three epitopes but rather weakly by MAb 904/6 to epitope 3. These studies suggest that epitope 1 of the M1 protein resides between amino acids 8 and 89, whereas epitopes 2 and possibly 3 are located between amino acids 89 and 141 or somewhat more carboxy distal. The intact M1 protein and its N-terminal 9- and 10-kilodalton peptides generated by formic acid or V8 protease cleavage, respectively, reconstituted with dipalmitoylphosphatidylcholine vesicles, but these N-terminal peptides had little effect on in vitro transcription of the RNP core. In sharp contrast, both intact M1 protein and the C-terminal 15-kilodalton formic acid fragment were able to inhibit viral transcription markedly. Moreover, MAb 289/4 (to epitope 2) reversed this inhibited transcription significantly. These studies suggest that the lipid-binding domain of the M1 protein is located within the amino-terminal third, whereas the site involved in the interaction of the M1 protein with RNP cores is located within the carboxy-terminal two-thirds.

Amino Acid Sequence↗

Structure-activity relationship of ghrelin: pharmacological study of ghrelin peptides.

Ghrelin, a novel peptide purified from the stomach, is the endogenous ligand of the growth hormone secretagogue receptor. The Ser(3) residue of ghrelin is modified with a lipid n-octanoic acid, a modification necessary for hormonal activity. To clarify the role of acyl modification and to identify the active core of ghrelin, we examined the activities of partially digested ghrelin and synthetic ghrelin derivatives. The activities confirmed that the N-terminal portion is the active core. Moreover, synthetic ghrelin derivatives demonstrated that octanoic acid is not the only modification of the Ser(3) side chain to sustain the activity of ghrelin; other acyl acid modifications maintained activity. Amino acid replacement of Ser(3) indicated that an L-configuration of the third residue is critical for ghrelin activity. In addition, more stable ether or thioether bonds are capable of replacing the octanoyl ester bond in ghrelin, advantageous for the generation of pharmaceuticals with longer stability.

Amino Acid Sequence↗

Towards abolition of immunogenic structures in insect cells: characterization of a honey-bee (Apis mellifera) multi-gene family reveals both an allergy-related core alpha1,3-fucosyltransferase and the first insect Lewis-histo-blood-group-related antigen-synthesizing enzyme.

Glycoproteins from honey-bee (Apis mellifera), such as phospholipase A2 and hyaluronidase, are well-known major bee-venom allergens. They carry N-linked oligosaccharide structures with two types of alpha1,3-fucosylation: the modification by alpha1,3-fucose of the innermost core GlcNAc, which constitutes an epitope recognized by IgE from some bee-venom-allergic patients, and an antennal Lewis-like GalNAcbeta1,4(Fucalpha1,3)GlcNAc moiety. We now report the cloning and expression of two cDNAs encoding the relevant active alpha1,3-FucTs (alpha1,3-fucosyltransferases). The first sequence, closest to that of fruitfly (Drosophila melanogaster) FucTA, was found to be a core alpha1,3-FucT (EC 2.4.1.214), as judged by several enzyme and biochemical assays. The second cDNA encoded an enzyme, most related to Drosophila FucTC, that was shown to be capable of generating the Le(x) [Galbeta1-4(Fucalpha1-3)GlcNAc] epitope in vitro and is the first Lewis-type alpha1,3-FucT (EC 2.4.1.152) to be described in insects. The transcription levels of these two genes in various tissues were examined: FucTA was found to be predominantly expressed in the brain tissue and venom glands, whereas FucTC transcripts were detected at highest levels in venom and hypopharyngeal glands. Very low expression of a third homologue of unknown function, FucTB, was also observed in various tissues. The characterization of these honey-bee gene products not only accounts for the observed alpha1,3-fucosylation of bee-venom glycoproteins, but is expected to aid the identification and subsequent down-regulation of the FucTs in insect cell lines of biotechnological importance.

Allergens↗

Overexpression and purification of a recombinant chimeric HIV type 2/HIV type 1 envelope peptide and application in an accelerated immunobased HIV type 1/2 antibody detection system (AIBS): a new rapid serological screening assay.

A chimeric HIV-2/HIV-1 envelope sequence containing an immunodominant region of HIV-2 gp36 and the corresponding region of HIV-1 gp41 was constructed and overexpressed in Escherichia coli. The recombinant product (rp21/18) was purified and applied in a novel antibody-screening assay. Characteristics in the design of this new principle are as follows: (1) the overall assay time is about 30 min; (2) the assay procedure includes three manipulation steps; and (3) the test shows a reliable performance with respect to sensitivity and specificity. The diluted serum sample and the protein G-horseradish peroxidase conjugate are added simultaneously into a coated (hybrid antigen HIV-1/2) and blocked microtiter plate well. The in-batch incubation of serum sample with protein G-horseradish peroxidase saves two manipulation steps that are normally necessary in the five-step procedure of a classical ELISA. AIBS was evaluated with commercially available seroconversion panels and with random negative serum samples from a blood bank. Seroconversion results demonstrated that AIBS has equivalent sensitivity to ELISAs and the third generation assays. The specificity was determined on a total blood donor population of 5012 (Red Cross Vienna, Austria). The repeat reactive rates for donor population were 0.02%. AIBS represents a general immunometric system (not only HIV antibodies). The entire assay procedure of AIBS evaluated for HIV-1/2 screening, including result reporting, can be performed automatically by several commercially available systems. Depending on these systems AIBS is potentially useful in laboratories or blood banks that have both high- and low-volume testing.

Amino Acid Sequence↗

Most peripheral B cells in mice are ligand selected.

Using amplified cDNA and genomic libraries, we have analyzed the VH gene repertoire of pre-B cells and various B cell subsets of conventional mice at the level of VH genes belonging to the J558 VH gene family. The sequence data were evaluated on the basis of a newly established list of 67 J558 VH genes that comprise approximately two-thirds of the J558 VH genes of the murine IgHb haplotype. The results of the analysis demonstrate that VH gene utilization in pre-B cells, although biased to some extent by B cell autonomous VH gene selection, scatters over the whole range of J558 VH genes present in the germline. In contrast, in mature, peripheral B cells comprising long-lived mu + delta high B cells as well as Ly-1 B cells, small overlapping sets of germline VH genes are dominantly expressed. The data indicate that the recruitment of newly generated B cells into the long-lived peripheral B cell pool is mediated through positive selection by internal and/or external antigens. Because of the absence of immunoglobulin class switching and somatic hypermutation, this process is different from the selection of memory B cells in T cell-dependent immune responses.

Animals↗

Structure of rearranged and germ-line rabbit V kappa genes indicated that the CDR3 is encoded by the V kappa gene.

The third complementary-determining regions (CDR3) of rabbit kappa chains are unusually long and the length is more heterogeneous when compared to those of the mouse and the human kappa chains. To study how the rabbit kappa light (L) chain genes create diversity and generate CDR3, we analyzed the structure of a rearranged variable kappa gene (Vr) and the variable (Vg) and joining (Jg) regions of the putative precursor genes. Alignment of the Vr gene sequence with that of the Vg and Jg regions allowed precise determination of the recombination event. Five nucleotides between the recombination point and the J2 heptamer were deleted, indicating flexibility in the recombination producing rabbit kappa chains. The entire Vg is contained in the rearranged product demonstrating that neither a D element nor an N sequence addition are required for the CDR3 formation. Comparison of the Vr and the Vg gene sequences show base substitutions suggesting that somatic mutations may contribute to rabbit kappa L chain diversity.

Amino Acid Sequence↗

Structure and genomic organization of the human B1 receptor gene for kinins (BDKRB1).

Two subtypes of mammalian bradykinin receptors, B1 and B2 (BDKRB1 and BDKRB2), have been defined based on their pharmacological properties. The B1 type kinin receptors have weak affinity for intact BK or Lys-BK but strong affinity for kinin metabolites without the C-terminal arginine (e.g., des-Arg9-BK and Lys-des-Arg9-BK, also called des-Arg10-kallidin), which are generated by kininase I. The B1 receptor expression is up-regulated following tissue injury and inflammation (hyperemia, exudation, hyperalgesia, etc.). In the present study, we have cloned and sequenced the gene encoding human B1 receptor from a human genomic library. The human B1 receptor gene contains three exons separated by two introns. The first and the second exon are noncoding, while the coding region and the 3'-flanking region are located entirely on the third exon. The exon-intron arrangement of the human B1 receptor gene shows significant similarity with the genes encoding the B2 receptor subtype in human, mouse, and rat. Sequence analysis of the 5'-flanking region revealed the presence of a consensus TATA box and of numerous candidate transcription factor binding sequences. Primer extension experiments have shown the existence of multiple transcription initiation sites situated downstream and upstream from the consensus TATA box. Genomic Southern blot analysis indicated that the human B1 receptor is encoded by a single-copy gene.

Animals↗

Mechanisms of homology-facilitated illegitimate recombination for foreign DNA acquisition in transformable Pseudomonas stutzeri.

Intra- and interspecific natural transformation has been observed in many prokaryotic species and is considered a fundamental mechanism for the generation of genetic variation. Recently, it has been described in detail how, in transformable Acinetobacter BD413 and Streptococcus pneumoniae, long stretches of nucleotides lacking homology were integrated into recipient genomes when they were linked on one side to a small piece of DNA with homology to resident DNA serving as a recA-dependent recombination anchor. Now, such homology-facilitated illegitimate recombination (HFIR) has also been detected in transformable Pseudomonas stutzeri. However, analysis of the recombinants revealed qualitative and quantitative differences in their generation compared with that in Acinetobacter BD413. In P. stutzeri, foreign DNA with an anchor sequence was integrated 105- to 106-fold less frequently than fully homologous DNA, but still at least 200-fold more frequently than without the anchor. The anchor sequence could be as small as 311 bp. Remarkably, in 98% of the events, the 3' end was integrated within the homologous anchor, whereas the 5' end underwent illegitimate fusion. Moreover, about one-third of the illegitimate fusion sites shared no or only a single identical basepair in foreign and resident DNA. The other fusions occurred within microhomologies of up to 6 bp with a higher GC content on average than the interacting nucleotide sequences. Foreign DNA of 69-1903 bp was integrated, and resident DNA of 22-2345 bp was lost. In a recA mutant, HFIR was not detectable. The findings suggest that genomic acquisition of foreign DNA by HFIR during transformation occurs widely in prokaryotes, but that details of the required recombination and strand fusion mechanisms may differ between organisms from different genera.

Base Sequence↗

Systematic and fully automated identification of protein sequence patterns.

We present an efficient algorithm to systematically and automatically identify patterns in protein sequence families. The procedure is based on the Splash deterministic pattern discovery algorithm and on a framework to assess the statistical significance of patterns. We demonstrate its application to the fully automated discovery of patterns in 974 PROSITE families (the complete subset of PROSITE families which are defined by patterns and contain DR records). Splash generates patterns with better specificity and undiminished sensitivity, or vice versa, in 28% of the families; identical statistics were obtained in 48% of the families, worse statistics in 15%, and mixed behavior in the remaining 9%. In about 75% of the cases, Splash patterns identify sequence sites that overlap more than 50% with the corresponding PROSITE pattern. The procedure is sufficiently rapid to enable its use for daily curation of existing motif and profile databases. Third, our results show that the statistical significance of discovered patterns correlates well with their biological significance. The trypsin subfamily of serine proteases is used to illustrate this method's ability to exhaustively discover all motifs in a family that are statistically and biologically significant. Finally, we discuss applications of sequence patterns to multiple sequence alignment and the training of more sensitive score-based motif models, akin to the procedure used by PSI-BLAST. All results are available at httpl//www.research.ibm.com/spat/.

Algorithms↗

Peculiarities of PAPA syndrome.

OBJECTIVES: To describe a family from New Zealand with the pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome, an autoinflammatory, variably expressed, erosive destructive form of arthritis. METHODS: Information was gained through medical records and interviews of the affected patients and wider family. DNA sequencing was performed at Seay Center for Musculoskeletal Research Texas Scottish Rite Hospital for Children. RESULTS: Five patients were affected over three generations with an E250Q mutation found on the CD2BP1 gene on chromosome 15. Common features included a severe, pauciarticular-onset, destructive peripheral arthritis, beginning at ages 5, 5, 2, 3 and 1(1/2) years. This combination marked cervical ankylosis (in two members), micrognathia and a more severe phenotype is unique. A third-generation family member treated early with DMARDs is following a less severe course. The skin involvement was variable, all with degrees of acne from puberty, though only one patient displayed pyoderma gangrenosum. A clear pattern of the arthritis switching off in adolescence and the triggering of skin disease was observed. CONCLUSIONS: Differing degrees of joint destruction, and cervical ankylosis in this family with the E250Q mutation demonstrate PAPA syndrome's variable expression. Further understanding of this rare condition and its pathway may allow better targeting of treatments, not just for families with this specific syndrome but also for other, more common, forms of arthritis.

Acne Vulgaris↗

Isolation of the MHC genes encoding the tumour-specific class I antigens expressed on a murine fibrosarcoma.

The C3H UV-induced fibrosarcoma, 1591, is highly immunogenic and, therefore, is readily rejected when transplanted into immunocompetent syngeneic recipients. Previous analysis of 1591 with tumour-specific or H-2-reactive monoclonal antibodies revealed that this antigenicity might be due to the expression of two novel class I major histocompatibility complex (MHC) antigens. In this report we describe the molecular cloning and initial characterization of three genes which account for all of the unique serological class I reactivities observed on this tumour. These include two distinct, but highly conserved, H-2L-like genes, and a third gene the product of which bears determinants which are characteristic of both the tumour and of class I products of the H-2k haplotype. Moreover, each of these genes contains a polymorphic restriction enzyme fragment which is detected in the class I sequences of 1591 relative to normal C3H tissue. Since the expression of these polymorphic class I sequences is relevant to the immunogenicity of 1591, the mutational events by which these genes were generated may be significant to the immunobiology of this tumour.

Animals↗

Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat.

The gene coding for the Na+/Ca2+ exchanger NCX1 is characterized by a cluster of six exons (A, B, C, D, E, and F) coding for a variable region in the COOH terminus of the large intracellular loop of the protein. Alternative splicing of these exons generates multiple tissue-specific variants of NCX1. Using reverse transcriptase-polymerase chain reaction, we analyzed eight previously described and four new splicing isoforms of NCX1 in a wide variety of tissues and cells. Exons A and B are mutually exclusive, as shown in earlier studies, and splicing isoforms containing exon A are preferentially expressed in heart, brain, and skeletal muscle, whereas splicing variants with exon B are found in all rat tissues except heart. The second and third isoforms of the Na+/Ca2+ exchanger, NCX2 and NCX3, show a deletion of 37 amino acids in the intracellular loop corresponding to parts of the variable region of NCX1. We identified three splicing isoforms of NCX3 in brain and skeletal muscle by reverse transcriptase-polymerase chain reaction. These splice variants are generated by including either of two alternative exons equivalent to the NCX1 exon A or B and by including or excluding a sequence equivalent to the NCX1 exon C. We did not detect any alternative splicing of NCX2. We examined selected tissues from neonatal and adult rats and found developmental regulation for NCX1 and NCX3 splicing isoforms in skeletal muscle. Specific isoform patterns were also detected for NCX1 and NCX3 in cultured cortical neurons, astrocytes, and oligodendrocytes. We suggest a new terminology to distinguish the different splice variants of individual NCX isoforms.

Aging↗

Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides.

The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.

Amino Acid Sequence↗

Complex events in the evolution of the haptoglobin gene cluster in primates.

Southern blot analyses of genomic DNA show that new world monkeys have only one haptoglobin gene but that chimpanzees, gorillas, orangutans, and old world monkeys have three. Humans have two: haptoglobin (Hp) and haptoglobin-related (Hpr). These observations suggest that a triplication of the haptoglobin locus occurred after the divergence of the new world monkeys, followed by a deletion of one locus in humans. To investigate these events, we have cloned the haptoglobin gene cluster in chimpanzee. The organization of the Hp and Hpr genes in chimpanzees is the same as in humans, including a retrovirus-like sequence in the first intron of Hpr. The third gene, which we name Hpp for haptoglobin primate, is 16 kilobases downstream of Hpr. A second copy of the retrovirus-like sequence occurs between Hpr and Hpp. The nucleotide sequence of the chimpanzee Hpp gene suggests that it may code for a functional protein, but the chimpanzee Hpr gene has a single base deletion in exon 5 that causes a frameshift. Comparison of the human and chimpanzee sequences suggests that the human Hpr gene was generated by a homologous unequal crossover between ancestral Hpr and Hpp genes. The crossover point lies within a 1.3-kilobase region containing exon 5 and 500 nucleotides 3' to the genes, but the exact point is obscured by a subsequent gene conversion event.

Animals↗

Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene.

The herpes simplex virus genome consists of two components, L and S, that invert relative to each other to yield four isomeric arrangements, prototype (P), inversion of the S component (Is), inversion of the L component (Il), and inversion of both components (Isl). Previous studies have shown that the 500-base-pair a sequences flanking the two components contain a cis-acting site for inversion. In an attempt to insert a third copy of the alpha 4 gene, the major regulatory gene mapping in the repeats flanking the S component, a fragment containing the alpha 4 gene and an origin of DNA synthesis, was recombined into the thymidine kinase gene mapping in the unique sequences of the L component. The resulting recombinants showed massive rearrangements and deletions mapping in the S component and in the junction between the L and S components. One recombinant (R7023) yielded two isomeric DNA arrangements, a major component consisting of Is and a minor component consisting of Isl. In these arrangements, the genome lacked the gene specifying glycoprotein E and all contiguous genes located between it and the alpha 0 gene in the inverted repeats of the L component. Among the deleted sequences were those encoding an origin of viral DNA synthesis, the alpha 47 gene, and the a sequences located at the junction between the L and S-components. The recombinant grew well in rabbit skin, 143TK-, and Vero cell lines. We conclude that the four unique genes deleted in R7023 are not essential for the growth of herpes simplex virus, at least in the cell lines tested, and that the b sequence of the inverted repeats of the L component also contains cis-acting sites for the inversion of herpes simplex virus DNA sequences.

Animals↗

Identification of a Manduca sexta NSF ortholog, a member of the AAA family of ATPases.

Transport between intracellular compartments requires the activity of an N-ethylmaleimide-sensitive fusion protein (NSF). NSF is a member of a growing family of ATPases regulating several membrane fusion reactions. We have cloned the NSF ortholog from the moth, Manduca sexta (MsNSF). MsNSF is highly conserved in domains critical for NSF function in vertebrates. MsNSF codes for a protein of 745 amino acids, translating to a M(r) of 83kDa in vitro. MsNSF is 72% and 61% similar in amino acid sequence to Drosophila and vertebrate NSFs, respectively. We expressed the D1 ATP domain of MsNSF toward which antibodies selective to MsNSF were generated. Affinity purified alpha-MsNSF antibodies detect a 83kDa protein which is highly enriched in nervous tissues. Levels of MsNSF expression are substantially lower in other tissues examined. Anti-MsNSF antibodies are capable of inhibiting vertebrate intra-Golgi transport of a cargo protein in vitro. The identification of NSF ortholog from Manduca, whose neuroendocrine system is well studied, should facilitate isolation of complexes involved in protein trafficking from insect models. Phylogenetic analysis of NSF and related proteins suggests that the members of the AAA family arose from different ancestors, since the ingroup was not monophyletic. Proteasomal subunits and p97 homologs form two distinct subfamilies, while NSF homologs branch in to the third.

Adenosine Triphosphatases↗

A DNA microarray for identification of virulence and antimicrobial resistance genes in Salmonella serovars and Escherichia coli.

Characterization of antimicrobial resistance and virulence gene profiles provides important information on the potential pathogenicity of bacteria. This information can be used to facilitate prompt and effective treatment of bacterial infections. We developed and tested a PCR-based microarray platform for detecting virulence and antimicrobial resistance genes in Salmonella serovars and Escherichia coli. Twelve Salmonella and seven E. coli isolates were screened for the presence of 25 virulence and 23 antimicrobial resistance genes. All S. Typhimurium DT104 isolates harbored virulence plasmids. E. coli O157:H7 isolates possessed virulence genes typical of enterohemorrhagic E. coli (EHEC), whereas E. coli O126 isolates contained virulence genes characteristic of enteropathogenic E. coli (EPEC) and E. coli O111, O78 and O147 isolates had virulence genes characteristic of enterotoxigenic E. coli (ETEC). Correlation between antimicrobial resistance phenotype and genotype was observed for each isolate. The aadA, tetA, and sulI genes were most commonly detected in bacteria resistant to streptomycin, tetracycline and sulfonamide, respectively. All isolates exhibiting resistance to third generation cephalosporins harbored the bla(CMY-2) and bla(TEM-1) genes. Microarray analysis is an effective method to rapidly screen Salmonella and E. coli for multiple antimicrobial resistance and virulence genes.

DNA, Bacterial↗

Energy transduction in lactic acid bacteria.

In the discovery of some general principles of energy transduction, lactic acid bacteria have played an important role. In this review, the energy transducing processes of lactic acid bacteria are discussed with the emphasis on the major developments of the past 5 years. This work not only includes the biochemistry of the enzymes and the bioenergetics of the processes, but also the genetics of the genes encoding the energy transducing proteins. The progress in the area of carbohydrate transport and metabolism is presented first. Sugar translocation involving ATP-driven transport, ion-linked cotransport, heterologous exchange and group translocation are discussed. The coupling of precursor uptake to product product excretion and the linkage of antiport mechanisms to the deiminase pathways of lactic acid bacteria is dealt with in the second section. The third topic relates to metabolic energy conservation by chemiosmotic processes. There is increasing evidence that precursor/product exchange in combination with precursor decarboxylation allows bacteria to generate additional metabolic energy. In the final section transport of nutrients and ions as well as mechanisms to excrete undesirable (toxic) compounds from the cells are discussed.

Adenosine Triphosphate↗