Complete nucleotide sequence of the genome organization of RNA2 of patchouli mild mosaic virus, a new favavirus

The complete nucleotide sequence and the genome organization of the RNA 2 of a patchouli mild mosaic virus (PaMMV) was determined. The sequence consists of 3591 nucleotides and contains a single long open reading frame sufficient to code for 118 K protein. Three proteins of 52 K, 44 K and 22 K could be encoded by the PaMMV RNA 2 genome. Our analysis of the N-terminal sequences of two species of coat protein (CP) allowed precise location of the CP cistrons within the polyprotein. 44 K and 22 K proteins are the coat proteins. The positions of the cleavage sites are Gln/Ala between 44 K and 22 K coat proteins and Gln/Gly between 52 K and 44 K proteins. Comparison of PaMMV RNA 2 with comoviral and nepoviral RNA 2 showed no sequence similarity. These results as well as previous serological studies strongly suggest that PaMMV is a member in the genus Fabavirus.     

The nucleotide sequence and genome organization of mushroom bacilliform virus: a single-stranded RNA virus of Agaricus bisporus (Lange) Imbach

Mushroom bacilliform virus (MBV) is found in association with spherical virus-like particles in cultivated mushrooms (Agaricus bisporus) afflicted with La France disease. MBV possesses a monopartite ssRNA genome of positive sense and differs from the majority of characterized mycoviruses, which contain segmented dsRNA genomes. We have cloned and sequenced the MBV genome and determined that its length is 4009 nucleotides. The MBV genome contains four major and three minor open reading frames and has 5' and 3' noncoding regions of 60 and 250 nucleotides, respectively. The putative RNA-dependent RNA polymerase and the coat protein display homology with corresponding proteins encoded by certain plant viruses, particularly luteoviruses and carmoviruses.     

Genomic organization and complete nucleotide sequence of the human PWP2 gene on chromosome 21

The human PWP2 gene is the human homologue of the yeast periodic tryptophan protein 2 (PWP2) gene and is a member of the gene family that contains tryptophan-aspartate (WD) repeats. Genomic sequencing revealed that the human PWP2 gene consists of 21 exons spanning approximately 24 kb and locates just between the two genes EHOC-1 and KNP-I and distal to a NotI site of LJ104 (D21S1460) on chromosome 21q22.3. Analysis of the 5'-flanking DNA sequence revealed that the upstream region of the PWP2 gene is associated with a CpG island containing the NotI site of LJ104. Since PWP2 is considered to be a candidate for genetic disorders mapped in the 21q22.3 region, the information including nucleotide sequence and genomic organization of the PWP2 gene should be invaluable for the mutation analysis of the corresponding genetic disorders.     

The complete nucleotide sequence of cosmid vector pTL5: location and origin of its genetic components

We investigated the incidence of peripheral neuropathy in 142 consecutive patients receiving high-dose etoposide between August 1988 and December 1991. A retrospective chart review was conducted. Six patients with hematologic malignancies presented with a new sensory polyneuropathy after receiving an intensive therapy regimen consisting of etoposide 60 mg/kg i.v. and melphalan 140-160 mg/m2 i.v. followed by autologous bone marrow transplantation. Neurologic symptoms began 2-8 weeks after transplant, were grade 2-3 in severity and pursued a chronic course with slow improvement over months. Electromyographic studies in three of the patients tested confirmed distal sensory polyneuropathy. All 6 patients had previously received vincristine 1-60 months prior to autotransplant. We conclude that peripheral neuropathy of moderate severity is a potential complication of high-dose etoposide therapy but appears to be self-limited.     

16S-23S and 23S-5S intergenic spacer regions of lactobacilli: nucleotide sequence, secondary structure and comparative analysis

Lactobacilli have been used as industrial starters for a long time, but in several cases their identification was, and still is, neither easy nor reliable. The aim of the present work was to examine whether the intergenic spacer regions could be of value in the identification of Lactobacillus species. For that purpose, the polymerase chain reaction (PCR) was used to amplify 16S-23S and 23S-5S spacer regions of Lactobacillus (L.) acidophilus, L. delbrueckii subsp. bulgaricus, L. casei, L. helveticus and L. curvatus. The PCR products were directly sequenced, and two forms of ribosomal RNA (rrn) operons were identified in each species studied: one with tandem tRNA(Ile)/tRNA(Ala) genes and the other one without tRNA genes. Our study revealed that the rrn operons of Lactobacillus species studied comprise the genes of 16S, 23S and 5S rRNA, in that order. Only the tRNA genes and the rRNA processing stems are highly conserved in spacer regions of lactobacilli. The divergence between the lactobacilli spacer region sequences arises from insertions and deletions of short sequences. These sequences could be interesting candidates for the development of species-specific probes. Theoretical RNA/RNA secondary structure models of the interaction between the two spacer region sequences were constructed. In conclusion, the two spacer region sequences may prove to be a useful alternative to 16S and 23S rDNA sequencing for designing species-specific probes and for establishing phylogenetic relationships between closely related species such as L. curvatus and L. casei or L. acidophilus and L. helveticus.     

The heat-shock gene hsp83 of Drosophila auraria: genomic organization, nucleotide sequence, and long antiparallel coupled ORFs (LAC ORFs)

The genomic organization of the hsp83 gene of Drosophila auraria, a far-eastern endemic species belonging to the montium subgroup of the melanogaster species group, is presented here. Based on in situ hybridization on polytene chromosomes, cDNA and genomic clone mapping, nucleotide sequencing, and genomic Southern analysis, hsp83 is shown to be present as a single-copy gene at locus 64B on the 3L chromosome arm in D. auraria. This gene is organized into two exons separated by a 929-bp intron. The first exon represents the mRNA leader sequence and is not translated, while the coding region, having a length of 2,151 bp, is solely included in the second exon. Nucleotide sequence comparisons of D. auraria hsp83 with homologous sequences from other organisms show high conservation of the coding region (88-92% identity) in the genus Drosophila, in addition to the conserved genomic organization of two-exons-one-intron, of comparable size and arrangement. A phylogenetic tree based on the protein sequences of homologous genes from representative organisms is in accord with the accredited phylogenetic position of D. auraria. In the hsp83 gene region, a second case of long antiparallel coupled open reading frames (LAC ORFs) for this species was found. The antiparallel to the hsp83 gene ORF is 1,554 bases long, while the two ORFs overlap has a size of 1,548 bp. The anti-hsp83 ORF does not show significant homology to any known gene sequences. In addition, no similar LAC ORF structures were found in homologous gene regions of other organisms.     

Primordial emergence of the recombination activating gene 1 (RAG1): sequence of the complete shark gene indicates homology to microbial integrases

The rearrangement of antibody and T-cell receptor gene segments is indispensable to the vertebrate immune response. All extant jawed vertebrates can rearrange these gene segments. This ability is conferred by the recombination activating genes I and II (RAG I and RAG II). To elucidate their origin and function, the cDNA encoding RAG I from a member of the most ancient class of extant gnathostomes, the Carcharhine sharks, was characterized. Homology domains identified within shark RAG I prompted sequence comparison analyses that suggested similarity of the RAG I and II genes, respectively, to the integrase family genes and integration host factor genes of the bacterial site-specific recombination system. Thus, the apparent explosive evolution (or "big bang") of the ancestral immune system may have been initiated by a transfer of microbial site-specific recombinases.     

Mouse liver nicotinamide N-methyltransferase: cDNA cloning, expression, and nucleotide sequence polymorphisms

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and structurally related compounds. We cloned mouse liver NNMT cDNA to make it possible to test the hypothesis that large differences among strains in levels of hepatic NNMT activity might be associated with strain-dependent variation in NNMT amino acid sequence. Mouse liver NNMT cDNA was 1015 nucleotides in length with a 792 nucleotide open reading frame (ORF) that was 83% identical to the nucleotide sequence of the human liver NNMT cDNA ORF. The mouse liver cDNA encoded a 264 amino acid protein with a calculated Mr value of 29.6 kDa. NNMT cDNA ORF sequences were then determined in five inbred strains of mice with very different levels of hepatic NNMT enzymatic activity. Although multiple differences among strains in nucleotide sequence were observed, none altered encoded amino acids. cDNA sequences for C57BL/6J and C3H/HeJ mice, prototypic strains with "high" and "low" levels of hepatic NNMT activity, respectively, were then expressed in COS-1 cells. Both expression constructs yielded comparable levels of enzyme activity, and biochemical properties of the expressed enzyme, including apparent Km values for substrates and IC50 values for inhibition by N1-methylnicotinamide, were very similar to those of mouse liver NNMT. Growth and development experiments were then conducted, which demonstrated that, although at 8 weeks of age average hepatic NNMT activity in C57BL/6J mice was 5-fold higher than that in C3H/HeJ mice, activities in the two strains were comparable by 30 weeks of age--indicating strain-dependent variation in the developmental expression of NNMT in mouse liver. These observations will serve to focus future studies of strain-dependent differences in murine hepatic NNMT on the regulation of the enzyme activity during growth and development.     

The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae

Previously, we have purified and characterized DNA helicase III from the yeast Saccharomyces cerevisiae [Shimizu, K. and Sugino, A. (1993) J. Biol. Chem. 268, 9578-9584]. Here, we have further characterized DNA helicase III activity. It was found that the combined action of the helicase III, yeast DNA topoisomerase I (yTop I), and yeast RPA protein on a covalently closed, circular DNA generates a highly underwound DNA species that has been called form I* or form U. Furthermore, these underwound structures can be accessed by yeast DNA polymerase I (alpha)-primase to initiate DNA synthesis. These reactions mimic in vivo initiation of chromosomal DNA replication. In order to clone the gene encoding DNA helicase III, a partial amino acid sequence of the purified DNA helicase III polypeptide was determined. Using a mix oligonucleotides synthesized based on the amino acid sequence of the helicase, we cloned the gene encoding the helicase III and found it to be identical to YER176W (HEL1) on chromosome V. The amino acid sequence predicted from the nucleotide sequence of the gene has conserved DNA helicase domains that are highly homologous to those of DNA helicases required for DNA replication. However, complete deletion of the gene from the chromosome did not result in any growth defect, suggesting that the gene product is not required for DNA synthesis or that it is functionally substituted by other helicase(s). Furthermore, the deletion strain does not exhibit sensitivity to any DNA-damaging reagents, although it is hypersensitive to calcofluor white, hygromycin, and papulacandin.     

Molecular hybridization of iodinated 4S, 5S, and 18S + 28S RNA to salamander chromosomes

4S, 5S, AND 18S + 28S RNA from the newt Taricha granulosa granulosa were iodinated in vitro with carrier-free 125I and hybridized to the denatured chromosomes of Taricha granulosa and Batrachoseps weighti. Iodinated 18S + 28S RNA hybridizes to the telomeric region on the shorter arm of chromosome 2 and close to the centromere on the shorter arm of chromosome 9 from T. granulosa. On this same salamander the label produced by the 5S RNA is located close to or on the centromere of chromosome 7 and the iodinated 4S RNA labels the distal end of the longer arm of chromosome 5. On the chromosomes of B. wrighti, 18S + 28S RNA hybridizes close to the centromeric region on the longer arm of the largest chromosome. Two centromeric sites are hybridized by the iodinated 5S RNA. After hybridization with iodinated 4S RNA, label is found near the end of the shorter arm of chromosome 3. It is concluded that both ribosomal and transfer RNA genes are clustered in the genome of these two salamanders.     

Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes

Synthetic coat protein complex I (COPI)-coated vesicles form spontaneously from large ( approximately 300 nm in diameter), chemically defined liposomes incubated with coatomer, Arf1p, and guanosine 5'-[gamma-thio]triphosphate. Coated vesicles are 40-70 nm in diameter, approximately the size of COPI vesicles formed from native membranes. The formation of COPI-coated buds and vesicles and the binding of Arf1p to donor liposomes depends on guanosine 5'-[gamma-thio]triphosphate. In contrast to the behavior of the COPII coat, coatomer binds to liposomes containing a variety of charged or neutral phospholipids. However, the formation of COPI buds and vesicles is stimulated by acidic phospholipids. In the absence of Arf1p, coatomer binds to liposomes containing dioleoylphosphatidic acid as a sole acidic phospholipid to form large coated surfaces without forming COPI-coated buds or vesicles. We conclude that Arf1p-GTP and coatomer comprise the minimum apparatus necessary to create a COPI-coated vesicle.     

Genetic organization, nucleotide sequence and regulation of expression of genes encoding phenol hydroxylase and catechol 1,2-dioxygenase in Acinetobacter calcoaceticus NCIB8250

Hapten refers to a chemical compound of small molar mass (typically less than 1000 daltons) that can bind with an antibody, but cannot initiate an immune response by itself unless it is conjugated to a protein carrier of larger molar mass. A novel method to prepare a hapten to generate anti-hapten immunity without covalent conjugation to a carrier was developed. Coating both water-soluble and -insoluble haptens onto a nitrocellulose membrane effectively presented haptens to the system and caused the generation of specific anti-hapten B lymphocytes and antibodies by immunization both in vitro and in vivo. This method has a potential to substitute for conventional hapten carrier conjugation to generate anti-hapten immunity.     

Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (pILL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli, the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the pILL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (pILL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in the chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (MBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.     

The recognition of protein structure and function from sequence: adding value to genome data

The explosion of DNA sequence data from genome projects presents many challenges. For instance, we must extend our current knowledge of protein structure and function so that it can be applied to these new sequences. The derivation of rules for the relationships between sequence and structure allow us to recognize a common fold by the use of tertiary templates. New techniques enable us to begin to meet the challenge of rule-based modelling of distantly related proteins. This paper describes an integrated and knowledge-based approach to the prediction of protein structure and function which can maximize the value of sequence information.