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PubMed1991

Intrageneric relationships of members of the genus Fusobacterium as determined by reverse transcriptase sequencing of small-subunit rRNA.

The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, "F. nucleatum subsp. fusiforme," and "F. nucleatum subsp. animalis"), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings.

Base Sequence↗
PubMed1997

Human telomerase contains evolutionarily conserved catalytic and structural subunits.

We have cloned and characterized a human gene encoding TP2 (telomerase-associated protein 2), a protein with similarity to reverse transcriptases and the catalytic telomerase subunits from Saccharomyces cerevisiae and Euplotes aediculatus. Indirect immunofluorescence revealed that TP2 was localized to the nucleus. Using antibodies to endogenous and epitope-tagged TP2, we found that TP2 was associated specifically with human telomerase activity and the recently identified telomerase-associated protein TP1. Mutation of conserved residues within the reverse transcriptase domain of TP2 severely reduced associated telomerase activity. These results suggest that telomerase is an evolutionarily conserved multisubunit complex composed of both structural and catalytic subunits.

Amino Acid Sequence↗
PubMed2003

Chimeric RNA/DNA oligonucleotide-based site-specific modification of the tobacco acetolactate syntase gene.

Single amino acid substitutions at either of two crucial positions in acetolactate synthase (ALS) result in a chlorsulfuron-insensitive form of this enzyme and, as a consequence, a herbicide-resistant phenotype. Here, we describe the successful in vivo targeting of endogenous tobacco (Nicotiana tabacum) ALS genes using chimeric RNA/DNA and all-DNA oligonucleotides at two different locations. Similar number of conversion events with two different chimeras indicates the absence of restricting influence of genomic target sequence on the gene repair in tobacco. Chlorsulfuron-resistant plants were regenerated from calli after mesophyll protoplast electroporation or leaf tissue particle bombardment with these specifically constructed chimeras. Sequence analysis and enzyme assays proved the resulting alterations to ALS at both DNA and protein levels. Furthermore, foliar application of chlorsulfuron confirmed the development of resistant phenotypes. Lines with proline-196-alanine, threonine, glutamine, or serine substitutions or with tryptophan-573-leucine substitutions were highly resistant at both cellular and whole plant levels, whereas lines with proline-196-leucine substitutions were less resistant. The stability of these modifications was demonstrated by the continuous growth of calli on chlorsulfuron-containing medium and by the transmission of herbicide resistance to progeny in a Mendelian manner. Ability of haploid state to promote chimera-mediated conversions is discussed.

Acetolactate Synthase↗
PubMed1995

CYP6B3: a second furanocoumarin-inducible cytochrome P450 expressed in Papilio polyxenes.

Cytochrome P450 monooxygenases in the larvae of Papilio polyxenes (black swallowtail) (Lepidoptera: Papilionidae) are capable of detoxifying linear and angular furanocoumarins found in their host plants. The CYP6B1 locus, which is transcriptionally induced in these larvae in response to xanthotoxin, encodes a P450 that principally metabolizes linear furanocoumarins such as xanthotoxin and bergapten. We have now cloned CYP6B3 cDNA derived from a second locus that is evolutionarily related to the CYP6B1 locus. Reverse transcription-PCR Southern analyses have demonstrated that CYP6B3 transcripts are expressed in response to a wider range of linear and angular furanocoumarins but at lower abundance than CYP6B1 transcripts. Whereas CYP6B1 transcripts are expressed at a low detectable level in uninduced control larvae and at high levels in xanthotoxin-induced larvae, CYP6B3 transcripts are nearly undetectable in control larvae and are highly induced by xanthotoxin and bergapten (linear furanocoumarins) as well as by angelicin and sphondin (angular furanocoumarins). The fact that these two CYP6B loci are differentially regulated by these four furanocoumarins indicates that P. polyxenes has adapted to the presence of the wide range of furanocoumarins in its host plants by diversifying its P450 isozyme structure and its furanocoumarin-responsive regulatory cascades.

Amino Acid Sequence↗
PubMed1992

Retron-Ec107 is inserted into the Escherichia coli genome by replacing a palindromic 34bp intergenic sequence.

Some natural isolates of Escherichia coli have been shown to produce a unique branched RNA-linked single-stranded DNA called msDNA. These bacteria contain a retro-element called retron consisting of the msr-msd region and the gene for reverse transcriptase (RT). All three E. coli retrons characterized to date have been shown to be integrated into a prophage or to be associated with phage-related genes. In this report, we identified a new msDNA from an E. coli wild strain. Using the msDNA as a probe, the retron for the msDNA was cloned and its DNA sequence was determined. The retron was found to consist of a 1.3kb DNA fragment, making it the smallest retron isolated to date. The msDNA produced from the retron consists of a 107 base single-stranded DNA, which is considered to be branched out from the 18th G residue of a 75-base RNA molecule by a 2',5'-phosphodiester linkage. Thus, the msDNA and the retron were designated msDNA-Ec107 and retron-Ec107, respectively. Most significantly, retron-Ec107 was inserted into the E. coli genome by replacing a 34bp intergenic sequence between the pyrE and ttk genes located at 82 min on the E. coli chromosome. Interestingly, the retron contains palindromic structures at both ends and the E. coli 34bp intergenic sequence also contains a 10bp inverted repeat structure. These palindromic structures might have played a role in the integration of retron-Ec107 into the E. coli genome.

Amino Acid Sequence↗
PubMed2002

rpoB mutation conferring rifampin resistance in Streptococcus pyogenes.

Streptococcus pyogenes BM4478 and Staphylococcus aureus BM4479 were isolated from a patient undergoing rifampin therapy. High-level resistance to rifampin was due to the following mutations in the rpoB gene: Ser522Leu in strain BM4478 and His526Asn and Ser574Leu in strain BM4479.

Amino Acid Sequence↗
PubMed1996

Role of FlgM in sigma D-dependent gene expression in Bacillus subtilis.

The alternative sigma factor sigma D directs transcription of a number of genes involved in chemotaxis, motility, and autolysis in Bacillus subtilis (sigmaD regulon). The activity of SigD is probably in contrast to that of FlgM, which acts as an antisigma factor and is responsible for the coupling of late flagellar gene expression to the assembly of the hook-basal body complex. We have characterized the effects of an in-frame deletion mutation of flgM. By transcriptional fusions to lacZ, we have shown that in FlgM-depleted strains there is a 10-fold increase in transcription from three different sigmaD-dependent promoters, i.e., Phag, PmotAB, and PfliDST. The number of flagellar filaments was only slightly increased by the flgM mutation. Overexpression of FlgM from a multicopy plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible spac promoter drastically reduced the level of transcription from the hag promoter. On the basis of these results, we conclude that, as in Salmonella typhimurium, FlgM inhibits the activity of SigD, but an additional element is involved in determining the number of flagellar filaments.

Bacillus subtilis↗
PubMed1989

The second-largest subunit of the poxvirus RNA polymerase is similar to the corresponding subunits of procaryotic and eucaryotic RNA polymerases.

We have characterized the poxvirus gene encoding the second-largest subunit of the viral DNA-dependent RNA polymerase. This gene, designated rpo132, is located in the HindIII A fragment of the DNA of the Brighton Red strain of cowpox virus. A similar gene is located in the corresponding position in the HindIII A fragment of the DNA of the Western Reserve strain of vaccinia virus. The rpo132 gene is transcribed throughout the viral multiplication cycle. It has two transcriptional start sites; one is operative at late times only, and the other (80 base pairs downstream) is operative both at early times and at late times. Neither early nor late transcripts originating from the latter RNA start site contain long 5'-terminal poly(A) sequences. The rpo132 gene has the capacity to encode primary gene products of two types. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 133-kilodalton (kDa) protein. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 132-kDa protein. Transcripts of the latter type are more abundant, suggesting that the 132-kDa protein is the major primary product of this gene. The predicted amino acid sequences of both gene products share extensive similarities with the amino acid sequences of the second-largest subunits of the following enzymes: the RNA polymerase of Escherichia coli, the RNA polymerase II of Saccharomyces cerevisiae, and the RNA polymerase II of Drosophila melanogaster. This result provides further evidence of relatedness between multisubunit DNA-dependent RNA polymerases.

Amino Acid Sequence↗
PubMed1998

The evolutionarily conserved eukaryotic arginine attenuator peptide regulates the movement of ribosomes that have translated it.

Translation of the upstream open reading frame (uORF) in the 5' leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control.

Amino Acid Sequence↗
PubMed1999

A comparative analysis of computational motif-detection methods.

The detection of motifs within and among families of protein sequences can provide useful information regarding the function, structure and evolution of a protein. With the increasing number of computer programs available for motif detection, a comparative evaluation of the programs from a biological perspective is warranted. This study uses a set of 20 reverse transcriptase (RT) protein sequences to test and compare the ability of 7 different computational methods to locate the ordered-series-of-motifs that are well characterized in the RT sequences. The results provide insight to biologists as to the usage, value, and reliability of the numerous methods available.

Algorithms↗
PubMed1997

Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins.

Sheath proteins designate low-molecular-weight non-amelogenin enamel polypeptides and their parent protein, which concentrate in the sheath space separating rod and inter-rod enamel (Uchida et al., 1995). Two porcine sheath proteins, with apparent molecular weights of 13 and 15 kDa, are characterized by protein sequencing. The primary structures of these polypeptides match a portion of the derived amino acid sequences of clones isolated from a porcine enamel organ epithelia-specific cDNA library. Sheath protein RNA messages differ by the inclusion or deletion of a 45-nucleotide segment and by the use of three alternative polyadenylation/cleavage sites. The secreted proteins are 395 and 380 residues in length, with molecular masses of 42,358 and 40,279 Daltons and calculated isoelectric points of 6.3 and 6.7, respectively. Polyclonal antibodies were raised against a synthetic peptide having the sheathlin-specific sequence EHETQQYEYSGGC. Immunohistochemistry with this antibody demonstrates that the protein encoded by the sheathlin cDNA is preferentially localized in the sheath space. We propose that the porcine sheath proteins and their proteolytic cleavage products be designated "sheathlin".

Amino Acid Sequence↗
PubMed1997

Insulin-like growth factor I in the teleost Oreochromis mossambicus, the tilapia: gene sequence, tissue expression, and cellular localization.

Using reverse transcription-PCR and molecular cloning, the complementary DNA sequence encoding preproinsulin-like growth factor I (IGF-I) of a teleost, the tilapia (Oreochromis mossambicus) was established from liver. At the amino acid level, tilapia IGF-I shows all residues necessary for the maintenance of tertiary structure and shares about 80% identity with IGF-I from other teleosts. The B and A domains of tilapia IGF-I show more than 90% homology to those of other teleosts and 86-93% to those of human. However, in contrast to salmonids, the C domain of tilapia is truncated. Reverse transcription-PCR analysis followed by Southern blotting with an internal probe specific for tilapia IGF-I indicated a transcript in liver, pancreas, gut, kidney, head kidney, gill, ovary, testis, eye, and brain. In correlation, parenchymal cells were identified as likely local production sites by the use of immunohistochemistry. IGF-I immunoreactivity was confined to D cells in pancreatic islets, gastroentero-endocrine cells, cells of renal proximal tubules, interrenal cells of the head kidney, gill chondrocytes, chloride cells of the gill epithelium, granulosa cells in the ovary, spermatocytes and Sertoli cells in testis, and neurons in retina and brain. The local production of IGF-I in multiple organs of the tilapia indicates paracrine/autocrine actions of IGF-I involved in organ-specific functions. The results further demonstrate that the primary structure of IGF-I, especially in the B and A domains, is highly conserved during phylogeny.

Amino Acid Sequence↗
PubMed1998

[Sequencing and homology analysis of cDNAs encoding FhGST and AsGST].

AIM: To study the homologies between FhGST, AsGST and Sj26GST. METHODS: Total RNAs were extracted from Fasciola hepatica (Fh) and Ascaris suum (As) by guanidine hydrochloride/CsCl ultra-centrifugation. Two pairs of primers were designed according to FhGST and AsGST sequences. RT-PCR was carried out using total RNA as template. PCR products were directly sequenced. Then the homology analysis was made using DNASIS software on nucleotide level. RESULTS: A FhGST403 bp fragment and a AsGST411 bp fragment were obtained and they both located in the open reading frame. CONCLUSION: On nucleotide level, the homologies are 59% and 54% between the two cloned fragments and Schistosoma japonicum 26 kDa GST (Sj26GST) respectively.

Animals↗
PubMed2005

Molecular taxonomy of a soil actinomycete isolate, KCCM10454 showing neuroprotective activity by 16S rRNA and rpoB gene analysis.

Epilepsy constitutes a significant public health problem, and even the newest drugs and neurosurgical techniques have proven unable to cure the disease. In order to select a group of isolates which could generate an active compound with neuroprotective or antiepileptic properties, we isolated 517 actinomycete strains from soil samples taken from Jeju Island, in South Korea. We then screened these strains for possible anti-apoptotic effects against serum deprivation-induced hippocampal cell death, using the 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay as an in vitro test. The excitotoxic glutamate analog, kainic acid (KA), was used to induce seizures in experimental mice in our in vivo tests. As a result of this testing, we located one strain which exhibited profound neuroprotective activity. This strain was identified as a Streptomyces species, and exhibited the rifampin-resistant genotype, Asn(AAC)442, according to the results of 16S rRNA and rpoB gene analyses.

Animals↗
PubMed1997

Cloning and expression pattern of a second [His5Trp7Tyr8]gonadotropin-releasing hormone (chicken GnRH-H-II) mRNA in goldfish: evidence for two distinct genes.

Complementary DNAs (cDNAs) encoding [Trp7Leu8]GnRH (sGnRH) and [His5Trp7Tyr8]GnRH (cGnRH-II) peptides have been isolated from the brain of goldfish (X. W. Lin and R. E. Peter, 1996, Gen. Comp. Endocrinol. 101, 282-296). In the present study we report the isolation of a second cDNA encoding cGnRH-II peptide in the brain of goldfish using reverse transcription (RT) and rapid amplification of cDNA ends. There is an overall 79.7% nucleotide sequence similarity between the two cGnRH-II cDNAs, with 65.3, 91.2, and 76.3% similarity between the 5'-untranslated regions, coding regions, and 3'-untranslated regions, respectively, of the two cGnRH-II cDNAs. Comparison of the two cGnRH-II precursors shows 87.2% amino acid similarity. The presence of two cGnRH-II genes was confirmed by the sequence analysis of the introns between exon II and exon III of the two cGnRH-II genes. Results indicate that the intron of the two cGnRH-II genes shows a high divergence in size and sequence, but contains the same splice junction. Expression of the two cGnRH-II mRNAs was detected by RT-polymerase chain reaction assay and Southern blot analysis in all five grossly dissected brain areas, olfactory bulbs and tracts, telencephalon, hypothalamus, optic tectum-thalamus, and posterior brain. However, there was a difference in apparent intensity of hybridization signal for the two cGnRH-II mRNAs in all brain areas, suggesting a difference of expression levels. sGnRH mRNA was detected in the olfactory bulbs, telencephalon, and hypothalamus, but not in midbrain and posterior brain areas. The present finding of duplicate cDNAs and genes for cGnRH-II in goldfish is in agreement with the recent tetraploidization in this species.

Amino Acid Sequence↗
PubMed2006

Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases.

Rpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large N-terminal helix and a short motif at the end of the module are critical in vivo. Lethal or conditional mutants of the C-terminal globe altered the binding of Rpb5 to Rpb1-beta25/26 (prolonging the Bridge helix) and Rpb1-alpha44/47 (ahead of the Switch 1 loop and binding Rpb5 in a two-hybrid assay). The large intervening segment of Rpb1 is held across the DNA Cleft by Rpb9, consistent with the synergy observed for rpb5 mutants and rpb9Delta or its RNA polymerase I rpa12Delta counterpart. Rpb1-beta25/26, Rpb1-alpha44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-alpha46/47 helix bundle were universally conserved. We conclude that the main function of the dual Rpb5-Rpb1 binding and the Rpb9-Rpb1 interaction is to hold the Bridge helix, the Rpb1-alpha44/47 helix bundle and the Switch 1 loop into a closely packed DNA-binding fold around the transcription bubble, in an organization shared by the two other nuclear RNA polymerases and by the archaeal and viral enzymes.

Amino Acid Sequence↗
PubMed1989

Transcription termination directed by heteroduplex thr attenuator templates. Evidence that the transcript stem and loop structure is the termination signal.

We have constructed both homoduplex and heteroduplex restriction fragment templates that contained mutations in the G + C-rich region of dyad symmetry in the Escherichia coli thr operon attenuator and have studied their termination properties in an in vitro transcription system. We found that the DNA sequence of the template strand determines the transcription termination efficiency. We also constructed two double mutant heteroduplex templates to examine the role of possible complementary base pairs between the nontranscribed DNA strand and the nascent RNA in the transcription bubble. There was no indication that the potential DNA-RNA interactions in the G + C-rich region are involved in transcription termination. Taken together, our results are consistent with and extend those obtained by Ryan and Chamberlin (Ryan, T., and Chamberlin, J. (1983) J. Biol. Chem. 258, 4690-4693) for the trp operon attenuator and argue strongly against models that propose that DNA hairpin formation in the nontranscribed DNA strand or pairing of the nascent RNA with the nontranscribed DNA strand are important for transcription termination.

Base Sequence↗