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PubMed1999

Regulation of human papillomavirus type 31 polyadenylation during the differentiation-dependent life cycle.

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed late in the differentiation-dependent life cycle from a promoter located in the E7 open reading frame (ORF) of the early region. These late HPV genes are transcribed by RNA polymerase II which reads through the region containing early polyadenylation signals and proceeds to a poly(A) site downstream of L1. In this study, we have investigated the mechanisms regulating differentiation-dependent polyadenylation and read-through in HPV-31. HPV-31 early transcripts were found to utilize a heterogeneous series of polyadenylation sites in undifferentiated cells. The sites for polyadenylation extended over a range of 100 nucleotides from within the E5 ORF to upstream of L2. Upon differentiation, the transcription of early genes increased, but no change in the heterogeneous distribution of 3' ends was detected. The early polyadenylation region was found to contain a single consensus hexanucleotide sequence, AAUAAA, as well as three weak binding sites for the cleavage stimulatory factor, CstF. In contrast to the heterogeneity at the early site, the 3' ends of late transcripts encoding L1 and L2 were localized to a narrow region downstream of the late AAUAAA element. The late polyadenylation signal was found to contain a single high-affinity site for CstF, as well as one consensus hexanucleotide sequence. By using a reporter assay, it was determined that the HPV-31 early polyadenylation sequences allowed significant levels of read-through into the late region in undifferentiated cells. Upon differentiation, this read-through was increased by approximately 50%, indicating that use of the early site decreased. Differentiation was also found to induce a 40% reduction in the levels of CstF subunits, which may contribute to the increased read-through of the early sequence. The insertion of the late high-affinity binding site for CstF into the early polyadenylation region significantly reduced the level of read-through, suggesting that these factors modulate read-through activity. Our studies demonstrate that HPV-31 late gene expression is regulated in a large part by posttranscriptional mechanisms, including the polyadenylation of early transcripts.

Binding Sites↗
PubMed1993

Two developmentally controlled promoters of Streptomyces coelicolor A3(2) that resemble the major class of motility-related promoters in other bacteria.

Experiments were designed to allow isolation of Streptomyces coelicolor promoters that depend on the whiG sporulation gene, which encodes a putative sigma factor important in the sporulation of aerial hyphae. The strategy, based on earlier evidence that sigma WhiG is limiting for sporulation (K. F. Chater, C. J. Burton, K. A. Plaskitt, M. J. Buttner, C. Méndez, and J. Helmann, Cell 59:133-143, 1989) was to seek DNA fragments that inhibit sporulation in aerial hyphae when present at a high copy number. In a suitable Sau3AI-generated library of DNA from S. coelicolor A3(2), two inserts were found to inhibit sporulation. Both inserts caused expression of the adjacent xylE reporter gene present in the vector in a developmentally normal strain of S. coelicolor, but there was no xylE expression in an otherwise isogenic whiG mutant. S1 nuclease protection experiments were done with RNAs isolated from these plasmid-bearing strains or from the wild-type strain lacking either recombinant plasmid. In each case, an apparent transcription start site was found upstream of an apparent open reading frame (ORF) and just downstream of sequences that resemble consensus features of promoters for motility-related genes in Bacillus subtilis and coliform bacteria. Such promoters depend on sigma factors (sigma D and sigma F, respectively) particularly similar to the deduced whiG gene product. Each of the putative whiG-dependent promoters is within an ORF that is upstream of, and potentially translationally coupled to, the putative whiG-dependent ORF (although use of one of the promoters would necessitate the use of a different start codon, further downstream). Thus, in unknown circumstances, the whiG-dependent ORFs may be expressed from a more remote promoter as part of a complex transcription unit.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of the vaccinia virus hemagglutinin gene.

The vaccinia virus hemagglutinin (HA) is a glycoprotein found on the plasma membrane of infected cells and the envelope of extracellular virus. Two forms of HA (85 and 68 kDa) are detected by immunoblot analysis. Although hemagglutination activity is only readily detectable late in infection, the 85-kDa HA appears early and accumulates throughout infection, whereas the 68-kDa form appears only late in the cycle. Production of the 68-kDa HA but not the 85-kDa HA was inhibited by either cytosine arabinoside or rifampin. Analysis of HA gene expression reveals a complex pattern of expression. The HA gene is transcribed early to yield a 1.65-kb dicistronic early transcript, consisting of the 945-bp HA open reading frame (ORF) fused to a 453-bp downstream ORF. Transcription from this site initiates 7 bases upstream of the AUG initiating codon of the HA ORF. Due to the discrepancy between the calculated size of the HA protein (33 kDa) and that reported for the unglycosylated HA protein derived from in vitro translation (58 kDa), we placed an early transcription termination signal (TTTTTAT) directly downstream of the 945-bp HA ORF. This led to a reduction in size of the early HA mRNA to 1.2 kb, as expected, but had no effect on the formation of either the 85- or 68-kDa protein. Transcripts originating from the early promoter are found throughout the infection cycle. However, after DNA replication, transcription from a second, late promoter ensues. The transcriptional start site of the late promoter is within a consensus TAAATG sequence located 135 bases upstream of the transcriptional start site of the first promoter. The late transcriptional start site is also found within an upstream ORF.

Amino Acid Sequence↗
PubMed1997

Squalene-hopene cyclase from Bradyrhizobium japonicum: cloning, expression, sequence analysis and comparison to other triterpenoid cyclases.

With the help of a PCR-based screening method, the gene encoding squalenehopene cyclase (SHC) of Bradyrhizobium japonicum USDA 110 was isolated from a cosmid library. The SHC catalyses the cyclization of squalene to hopanoids, a class of triterpenoid lipids recently discovered in nitrogen-fixing, root-nodule-forming Bradyrhizobium bacteria. Hybridization experiments showed that the gene is present in bacteria of all Bradyrhizobium strains tested and in photosynthetic bacteria forming stem nodules on tropical legumes of the genus Aeschynomene. The Bradyrhizobium shc gene is 1983 bp in length and encodes a protein of 660 amino acid residues with a calculated molecular mass of 73671 Da. Comparison of the deduced amino acid sequence with the sequences of other SHCs revealed highest similarity (70%) to the SHC from the Gram-negative Zymomonas mobilis and lower similarity (48%) to the SHCs from the Gram-positive Alicyclobacillus acidocaldarius and Alicyclobacillus acidoterrestris. Bradyrhizobium SHC also showed similarity (38-43%) to eukaryotic oxidosqualene cyclases. The B. japonicum shc gene was expressed in Escherichia coli. The recombinant SHC catalysed the cyclization of squalene to the hopanoids hopene and diplopterol in vitro. However, the formation of the gammacerane derivative tetrahymanol, which is produced in addition to hopanoids in B. japonicum strains in vivo, could not be detected in vitro. Therefore, the presence of a second squalene cyclase in B. japonicum can be assumed. Sequence analysis of 0.5 kb upstream from the shc gene identified a partial ORF with significant similarity to the C-terminus of an ORF located immediately upstream from the shc gene in Z. mobilis. Both ORFs also showed similarity to phytoene desaturases from cyanobacteria and plants. The 3'-end of this ORF from B. japonicum overlaps with 13 bp at the 5'-end of shc. The close proximity of this ORF to shc suggests that shc and this ORF may be part of an operon.

Amino Acid Sequence↗
PubMed1991

Characterization of an operon encoding succinyl-CoA synthetase and malate dehydrogenase from Thermus flavus AT-62 and its expression in Escherichia coli.

An open reading frame (ORF) was found upstream of the mdh gene in Thermus flavus by computer-aided analysis. It was identified as the gene encoding the alpha subunit of succinyl-CoA synthetase (SCS) and termed scsA. Nucleotide sequencing of a further upstream region revealed the presence of another ORF, corresponding to the sequence of the beta subunit of SCS. The latter gene was termed scsB. The scsB gene was found to contain an unusual translational initiation codon, TTG. S1 nuclease mapping indicates that transcription starts at the nucleotide at position--31 upstream of the initiation codon of the beta gene. The scsB and scsA genes along with the mdh gene appear to form an operon and are most likely co-transcribed in this order, because the intercistronic regions between them are very short; in fact, the termination codon of scsB overlaps the initiation codon of scsA. A stretch characteristic of the--10 region of a typical prokaryotic promoter was found upstream of scsB, whereas no sequence characteristic of a typical--35 region was present. Escherichia coli harboring a plasmid containing scsA and scsB did not produce thermostable SCS activity, even when a synthetic promoter for E. coli was attached. However, when an inverted repeat present in front of scsB, which covers the putative ribosome-binding site and is capable of forming a stable stem-loop structure, was altered by site-directed mutagenesis, overproduction of heat-stable SCS was observed.

Amino Acid Sequence↗
PubMed1990

The complete nucleotide sequence of PEBV RNA2 reveals the presence of a novel open reading frame and provides insights into the structure of tobraviral subgenomic promoters.

The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter.

Amino Acid Sequence↗
PubMed1991

Sequence comparison of the 5' end of mRNA 3 from transmissible gastroenteritis virus and porcine respiratory coronavirus.

Analysis of porcine transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) mRNA species indicated a deletion in mRNA 3 of PRCV. Polymerase chain reaction (PCR) was used to clone the 5' end of mRNA 3 from PRCV for comparison with the equivalent region in TGEV. Small deletions were observed within and around the PRCV sequence equivalent to the putative open reading frame (ORF) ORF-3a identified in TGEV. The potential RNA polymerase-leader complex binding site (leader RNA binding site), ACTAAAC, found upstream of ORF-3a in TGEV, was absent from the PRCV genome but a potential site was found in the PRCV genome upstream of a gene equivalent to TGEV ORF-3b. PCR analysis, using primers corresponding to sequences within the ORF-3b gene and the leader RNA sequence, confirmed that the leader RNA binding site was upstream of a gene equivalent to TGEV ORF-3b on PRCV mRNA 3 but upstream of ORF-3a on TGEV mRNA 3. The presence of the new leader RNA binding site would be responsible for generating the smaller mRNA 3 species found in PRCV-infected cells.

Amino Acid Sequence↗
PubMed1991

Organization and nucleotide sequences of two lactococcal bacteriocin operons.

Two distinct regions of the Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6, each of which specified bacteriocin production as well as immunity, have been sequenced and analyzed by deletion and frameshift mutation analyses. On a 1.8-kb ScaI-ClaI fragment specifying low antagonistic activity, three open reading frames (ORFs) were present, which were organized in an operon. The first two ORFs, containing 69 and 77 codons, respectively, were involved in bacteriocin activity, whereas the third ORF, containing 154 codons, was essential for immunity. Primer extension analysis indicated the presence of a promoter upstream of the ORFs. Two ORFs were present on a 1.3-kb ScaI-HindII fragment specifying high antagonistic activity. The first ORF, containing 75 codons, specified bacteriocin activity. The second ORF, containing 98 codons, specified immunity. The nucleotide sequences of both fragments upstream of the first ORFs as well as the first 20 bp of the first ORF of both bacteriocin operons appeared to be identical.

Amino Acid Sequence↗
PubMed2001

Promoters of the CATG-specific methyltransferase gene hpyIM differ between iceA1 and iceA2 Helicobacter pylori strains.

Helicobacter pylori strains can be divided into two groups, based on the presence of two unrelated genes, iceA1 and iceA2, that occupy the same genomic locus. hpyIM, located immediately downstream of either gene, encodes a functional CATG-specific methyltransferase. Despite the strong conservation of the hpyIM open reading frame (ORF) among all H. pylori strains, the sequences upstream of the ORF in iceA1 and iceA2 strains are substantially different. To explore the roles of these upstream sequences in hpyIM regulation, promoter analysis of hpyIM was performed. Both deletion mutation and primer extension analyses demonstrate that the hpyIM promoters differ between H. pylori strains 60190 (iceA1) and J188 (iceA2). In strain 60190, hpyIM has two promoters, P(a) or P(I), which may function independently, whereas only one hpyIM promoter, P(c), was found in strain J188. The XylE assay showed that the hpyIM transcription level was much higher in strain 60190 than in strain J188, indicating that regulation of hpyIM transcription differs between the H. pylori iceA1 strain (60190) and iceA2 strains (J188). Since the iceA1 and iceA2 sequences are highly conserved within iceA1 or iceA2 strains, we conclude that promoters of the CATG-specific methylase gene hpyIM differ between iceA1 and iceA2 strains, which leads to differences in regulation of hpyIM transcription.

Acetyltransferases↗
PubMed1997

Characterization of a cry2A gene cloned from an isolate of Bacillus thuringiensis serovar sotto.

A cry2A-type gene, designated as cry2(SKW), was cloned from Bacillus thuringiensis serovar sotto SKW01-10.2-06, and some unique features of the gene were revealed. The cry2(SKW) gene encoded a polypeptide of 635 residues with a predicted molecular mass of 71,137 Da. Cry2(SKW) had 95.4% identity with Cry2Aa in amino acid sequence and was two residues longer than Cry2Aa. Two open reading frames (ORFs), designated as orf1 and orf2, were present upstream of the cry2(SKW) and showed high homology with the corresponding ORFs in the cry2Aa operon. The Orf2 from SKW01-10.2-06 contained a region of repeated sequences. However, unlike Cry2Aa, Cry2(SKW) formed the cuboidal crystalline inclusions when the cry2(SKW) gene was expressed in an acrystalliferous B. thuringiensis strain in the absence of the upstream ORFs. Furthermore, Cry2(SKW) was less toxic to a lepidopteran species, Bombyx mori, than Cry2Aa in spite of high homology between the two proteins.

Amino Acid Sequence↗
PubMed1994

Sequence of the HAP3 transcription factor of Kluyveromyces lactis predicts the presence of a novel 4-cysteine zinc-finger motif.

The Kluyveromyces lactis homologue of the Saccharomyces cerevisiae HAP3 gene was isolated by functional complementation of the respiratory-deficient phenotype of the S. cerevisiae hap3::HIS4 strain SHY40. The KlHAP3 gene encodes a protein of 205 amino acids, of which the central B-domain of 90 residues is highly homologous to HAP3 counterparts of S. cerevisiae and higher eukaryotes. The protein contains a novel 4-cysteine zinc-finger motif and we propose by analogy that all other homologous HAP3 proteins contain the same motif, with the position containing the third cysteine being occupied by a serine residue. In contrast to the situation in S. cerevisiae, disruption of the KlHAP3 gene in K. lactis does not result in a respiratory-deficient phenotype and the growth of the null strain is indistinguishable from wild type. There is also no effect on the expression of the carbon source-regulated KlCYC1 gene, suggesting either a different role for the HAP2/3/4 complex, or the existence of a different mechanism of carbon source regulation. Sequence verification of the S. cerevisiae HAP3 locus reveals that, just as in K. lactis, a long open reading frame (ORF) is present upstream of the HAP3 gene. These highly homologous ORFs are predicted to have at least eight membrane-spanning fragments, but do not show significant homology to any known sequence present in databases. The ScORFX gene is transcribed in the opposite direction to ScHAP3, but, in contrast to an earlier report by Hahn et al. (1988), the transcripts of the two genes do not overlap. The model proposed by these authors, in which the ScHAP3 gene is regulated by an anti-sense non-coding mRNA, is therefore not correct.

Amino Acid Sequence↗
PubMed2005

Deletion of M2 gene open reading frames 1 and 2 of human metapneumovirus: effects on RNA synthesis, attenuation, and immunogenicity.

The M2 gene of human metapneumovirus (HMPV) contains two overlapping open reading frames (ORFs), M2-1 and M2-2. The expression of separate M2-1 and M2-2 proteins from these ORFs was confirmed, and recombinant HMPVs were recovered in which expression of M2-1 and M2-2 was ablated individually or together [rdeltaM2-1, rdeltaM2-2, and rdeltaM2(1+2)]. Each M2 mutant virus directed efficient multicycle growth in Vero cells. The ability to recover HMPV lacking M2-1 contrasts with human respiratory syncytial virus, for which M2-1 is an essential transcription factor. Expression of the downstream HMPV M2-2 ORF was not reduced when translation of the upstream M2-1 ORF was silenced, indicating that it is initiated separately. The rdeltaM2-2 mutants exhibited a two- to fivefold increase in the accumulation of mRNA, normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in hamsters. Animals infected intranasally with rdeltaM2-1 or rdeltaM2(1+2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 postinfection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with rdeltaM2-2, virus was recovered from only 1 of 12 animals and only in the nasal turbinates on a single day. However, all of the animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The HMPV rdeltaM2-2 virus is a promising and highly attenuated HMPV vaccine candidate.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the macromomycin apoprotein gene and its expression in Streptomyces macromomyceticus.

A 1.6 kb Sph I-Sac I DNA fragment from Streptomyces macromomyceticus, expected to include the macromomycin (MCM) apoprotein gene, was sequenced. The fragment (1,556 bp) was found to include a putative promoter, an ORF directing pre-apoprotein which should be split into the leader peptide (Met1 to Gly32) and the MCM apoprotein (Ala33 to Ala144), and a putative terminator. The amino acid sequence deduced from the base sequence of the DNA is consistent with the amino acid sequence previously determined by the Edman degradation and other procedures applied to the protein, except base sequence AAC coding for Asn was found rather than Asp111 previously reported. The GC content of the 3rd letters throughout the ORF was 92% in contrast to the sum of the first and the second letters, 62%. There was a low GC content stretch of 20 bp (30% GC) at about 120 bp upstream of the ORF. The Pst I-Sph I 620 bp fragment including the low GC content stretch showed promoter activity when subcloned in a promoter probe vector. About 700 nucleotides long mRNA, which is long enough to span the ORF and the bordering regions, was identified using the Northern blot analysis. A primer extension experiment showed that the transcriptional starting point was A at 89 bp upstream of the ORF. Dot blot analysis of expression of MCM apoprotein gene indicated that the gene was expressed nearly constitutively, while production of holo MCM (the complex consisting of MCM apoprotein and a specific chromophore) depended greatly on culture conditions.

Amino Acid Sequence↗
PubMed1990

The p6.5 gene region of a nuclear polyhedrosis virus of Orgyia pseudotsugata: DNA sequence and transcriptional analysis of four late genes.

The gene encoding the basic DNA-binding protein (p6.5) of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was localized by Southern blot analysis using a cDNA probe containing the Autographa californica virus (AcMNPV) p6.9 gene. The OpMNPV p6.5 gene was mapped to the HindIII G fragment at map unit 67. Nucleotide sequence and transcriptional analysis of a 3.26 kb region encompassing this area revealed four open reading frames (ORFs 1 to 4) oriented in the same direction. ORF 1 demonstrated a seven codon overlap with ORF 2. Messenger RNAs initiated upstream of each of the four ORFs late in infection and were coterminal at a single site downstream of the fourth ORF. The conserved late gene promoter/mRNA start site sequence (ATAAG) was present upstream of all the ORFs, but did not appear to be the major site of mRNA initiation for the third ORF, as determined by primer extension analysis. The fourth ORF in this series encoded a predicted peptide of 51 amino acids (6.5K), which was 80% similar to the p6.9 basic DNA-binding protein of AcMNPV.

Amino Acid Sequence↗
PubMed1999

Identification of a new promoter in the early region of the human papillomavirus type 16 genome.

Transcription of the human papillomavirus type 16 (HPV-16) genome is controlled by several promoters; the P(97) promoter is considered to be the main one. An additional promoter has been identified within the E7 ORF as well as an antisense promoter just upstream of the L2 ORF. The significance of these promoters for early and late gene expression and their activity related to cell differentiation is not known in detail. Identification of two new, previously undescribed transcription start sites at nt 542 just upstream of the E7 ORF and at nt 611 within the E7 ORF is reported. The promoter responsible for the start site at nt 542 (P(542)) was active in SiHa, HeLa and C33A cells. Very low promoter activity was found upstream of the nt 611 start site. The E7 protein has previously been shown to be synthesized from a polycistronic mRNA encoding both the E6 and E7 proteins under the control of the P(97) promoter. The data reported in the present paper suggest that promoter P(542) may control synthesis of the E7 oncoprotein from a monocistronic mRNA.

Base Sequence↗
PubMed1998

Characterization of the Escherichia coli RNA 3'-terminal phosphate cyclase and its sigma54-regulated operon.

The RNA 3'-terminal phosphate cyclase catalyzes the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of various RNA substrates. Recent cloning of a cDNA encoding the human cyclase indicated that genes encoding cyclase-like proteins are conserved among Eucarya, Bacteria, and Archaea. The protein encoded by the Escherichia coli gene was overexpressed and shown to have the RNA 3'-phosphate cyclase activity (Genschik, P., Billy, E., Swianiewicz, M., and Filipowicz, W. (1997) EMBO J. 16, 2955-2967). Analysis of the requirements and substrate specificity of the E. coli protein, presented in this work, demonstrates that properties of the bacterial and human enzymes are similar. ATP is the best cofactor (Km = 20 microM), whereas GTP (Km = 100 microM) and other nucleoside triphosphates (NTPs) act less efficiently. The enzyme undergoes nucleotidylation in the presence of [alpha-32P]ATP and, to a lesser extent, also in the presence of other NTPs. Comparison of 3'-phosphorylated oligoribonucleotides and oligodeoxyribonucleotides of identical sequence demonstrated that the latter are at least 300-fold poorer substrates for the enzyme. The E. coli cyclase gene, named rtcA, forms part of an uncharacterized operon containing two additional open reading frames (ORFs). The ORF positioned immediately upstream, named rtcB, encodes a protein that is also highly conserved between Eucarya, Bacteria, and Archaea. Another ORF, called rtcR, is positioned upstream of the rtcA/rtcB unit and is transcribed in the opposite direction. It encodes a protein having features of sigma54-dependent regulators. By overexpressing the N-terminally truncated form of RtcR, we demonstrate that this regulator indeed controls expression of rtcA and rtcB in a sigma54-dependent manner. Also consistent with the involvement of sigma54, the region upstream of the transcription start site of the rtcA/rtcB mRNA contains the -12 and -24 elements, TTGCA and TGGCA, respectively, characteristic of sigma54-dependent promoters. The cyclase gene is nonessential as demonstrated by knockout experiments. Possible functions of the cyclase in RNA metabolism are discussed.

Amino Acid Sequence↗
PubMed2002

The ribulose-1,5-bisphosphate carboxylase/oxygenase gene cluster of Methylococcus capsulatus (Bath).

The genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Methylococcus capsulatus (Bath) were localised to an 8.3-kb EcoRI fragment of the genome. Genes encoding the large subunit ( cbbL), small subunit ( cbbS) and putative regulatory gene ( cbbQ) were shown to be located on one cluster. Surprisingly, cbbO, a second putative regulatory gene, was not located in the remaining 1.2-kb downstream (3') of cbbQ. However, probing of the M. capsulatus (Bath) genome with cbbO from Nitrosomonas europaea demonstrated that a cbbO homologue was contained within a separate 3.0-kb EcoRI fragment. Instead of a cbbR ORF being located upstream (5') of cbbL, there was a moxR-like ORF that was transcribed in the opposite direction to cbbL. There were three additional ORFs within the large 8.3-kb EcoRI fragment: a pyrE-like ORF, an rnr-like ORF and an incomplete ORF with no sequence similarity to any known protein. Phylogenetic analysis of cbbL from M. capsulatus (Bath) placed it within clade A of the green-type Form 1 Rubisco. cbbL was expressed in M. capsulatus (Bath) when grown with methane as a sole carbon and energy source under both copper-replete and copper-limited conditions. M. capsulatus (Bath) was capable of autotrophic growth on solid medium but not in liquid medium. Preliminarily investigations suggested that other methanotrophs may also be capable of autotrophic growth. Rubisco genes were also identified, by PCR, in Methylococcus-like strains and Methylocaldum species; however, no Rubisco genes were found in Methylomicrobium album BG8, Methylomonas methanica S1, Methylomonas rubra, Methylosinus trichosporium OB3b or Methylocystis parvus OBBP.

Amino Acid Sequence↗
PubMed1995

Identification and characterization of the thymidine kinase gene of Yaba virus.

DNA of Yaba virus, a member of the poxviruses, was mapped by cross-hybridization between fragments of various restriction enzymes. The genome was approximately 135 kb in length and possessed two characteristic features of poxviruses: cross-links and inverted terminal repeats at both termini. Hybridization of fragments of Yaba virus DNA to known vaccinia virus DNA fragments indicated that the thymidine kinase (TK) gene mapped within the 0.9 kb XhoI-HincII fragment between 52.5 and 53.5 kb from the left end of the genome. The fragment could rescue the TK+ phenotype in TK- cells preinfected with a TK vaccinia virus mutant. Nucleotide sequencing of the fragment revealed an ORF capable of encoding 181 amino acids. The sequence TAAAAATGAAAAATTA upstream of the ORF was considered to be the promoter and the downstream sequence TTTTTAT to be the early transcription termination signal. These sequences are in good accord with the consensus regulatory sequences for the expression of early genes of other known poxviruses. The amino acid sequence similarity among the poxvirus TK genes suggests that Yaba virus is most closely related to swinepox virus and less similar to fowlpox virus.

Amino Acid Sequence↗