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PubMed2000

The 28S-18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor.

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from approximately 11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an approximately 55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A' pre-rRNA processing sites within the 5' external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5' ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C. fasciculata and Trypanosoma brucei involves 3'-terminal addition of three A residues that are not present in the corresponding DNA sequences.

Animals↗
PubMed1994

The human cytomegalovirus origin of DNA replication (oriLyt) is the critical cis-acting sequence regulating replication-dependent late induction of the viral 1.2-kilobase RNA promoter.

Plasmid constructs containing the 1.2-kb RNA promoter from the long terminal repeat region of human cytomegalovirus (HCMV) display the early-phase regulation of this promoter but lack the characteristic late induction (E. J. Wade, K. M. Klucher, and D. H. Spector, J. Virol. 66:2407-2417, 1992). To determine if the HCMV origin of replication (oriLyt) was necessary and sufficient for the late induction of the 1.2-kb RNA promoter, we cloned a 9.6-kbp segment of the origin of replication onto the p456 OCAT plasmid containing the 1.2-kb RNA promoter. This plasmid was designated ori456 OCAT. A control construct, which contains all of the same sequences as the ori456 OCAT construct except that a 2.4-kbp segment derived from HCMV EcoRI segment U is inverted in orientation to disrupt the origin function, was designated inv456 OCAT. After electroporation into human fibroblast cells and infection with HCMV 24 h later, ori456 OCAT replicated and showed the same early and late transcription pattern as the authentic viral 1.2-kb RNA. Under similar conditions, the inv456 OCAT neither replicated nor showed late induction. Experiments using plasmids synthesized in bacteria lacking methylation activity demonstrated that the late induction was not dependent on the change in methylation state of the plasmids. Ganciclovir, an inhibitor of the HCMV DNA polymerase, was used to demonstrate the replication dependence of the expression of the virally encoded 1.2-kb RNA, while the nearby early 2.7-kb RNA was unaffected. Ganciclovir also inhibited the late induction of the chloramphenicol acetyltransferase gene from ori456 OCAT, while expression from inv456 OCAT increased. Site-specific mutations in two previously identified important regulatory elements of the 1.2-kb RNA promoter, the AP1-binding site and the CATA site, indicated that these sites continue to contribute to promoter activity at late times but that the replication-dependent late induction acts independently of these sites. Possible mechanisms underlying the late induction are discussed.

Base Sequence↗
PubMed1986

Molecular cloning and restriction endonuclease mapping of the rat cytomegalovirus genome.

Rat cytomegalovirus (RCMV) DNA was cleaved by restriction endonuclease EcoRI into 24 fragments ranging in mol. wt. from 34 X 10(6) to 0.20 X 10(6), of which 18 fragments could be cloned in plasmid pACYC 184. Restriction endonuclease XbaI cleaved the RCMV genome into 28 fragments, ranging in size from 44 X 10(6) to 0.81 X 10(6), of which 24 fragments were cloned in plasmid pSP62-PL. Among the restriction fragments that could not be cloned were two major terminal colinear fragments, EcoRI-A (34 X 10(6)) and XbaI-A (44 X 10(6)). Thus, the complete sets of recombinant plasmids spanned about 70% of the RCMV genome. Our mapping results including determination of the termini of the genome, characterization of double digestion products of restriction fragments and cross-hybridization of 35S-labelled (cloned) EcoRI and XbaI fragments to Southern blots of EcoRI-, XbaI- or BglII-cleaved RCMV DNA, allowed us to construct the EcoRI and XbaI restriction maps of RCMV DNA. Since no cross-hybridization between internal fragments was seen, it is concluded that the RCMV genome consists of a long unique sequence of 224 kilobases without internal inverted repeat sequences, which is similar to the structures of murine and guinea-pig CMV DNA but unlike that of human CMV DNA. In a minor population (approx. 20%) of the RCMV DNA, one terminus was found to be larger by 0.35 X 10(6) mol. wt. The nature of this fragment is unclear at the moment.

Animals↗
PubMed1986

Rearrangements and insertions in the Moloney murine leukemia virus long terminal repeat alter biological properties in vivo and in vitro.

The effects of rearrangement and insertion of sequences in the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. The alterations were made by recombinant DNA manipulations on a plasmid subclone containing an M-MuLV LTR. Promoter activity of altered LTRs was measured by fusion to the bacterial chloramphenicol acetyltransferase gene, followed by transient expression assay in NIH 3T3 cells. M-MuLV proviral organizations containing the altered LTRs were also generated, and infectious virus was recovered by transfection. Infectivity of the resulting virus was quantified by XC plaque assay, and pathogenicity was determined by inoculating neonatal NIH Swiss mice. Inversion of sequences in the U3 region containing the tandemly repeated enhancer sequences (-150 to -353 base pairs [bp]) reduced promoter activity approximately fivefold in the transient-expression assays. Infectious virus containing the inverted sequences (Mo- M-MuLV) showed a 20-fold reduction in relative infectivity compared with wild-type M-MuLV, but the virus still induced thymus-derived lymphoblastic lymphoma or leukemia in mice, with essentially the same kinetics as for wild-type M-MuLV. We previously derived an M-MuLV which carried inserted enhancer sequences from the F101 strain of polyomavirus (Mo + PyF101 M-MuLV) and showed that this virus is nonleukemogenic. In Mo + PyF101 M-MuLV, the PyF101 sequences were inserted between the M-MuLV promoter and the M-MuLV enhancers (at -150 bp). A new LTR was generated in which the PyF101 sequences were inserted to the 5' side of the M-MuLV enhancers (at -353 bp, PyF101 + Mo M-MuLV). The PyF101 + Mo LTR exhibited promoter activity similar (40 to 50%) to that of wild-type M-MuLV, and infectious PyF101 + Mo M-MuLV had high infectivity on NIH 3T3 cells (50% of wild type). In contrast to the nonleukemogenic Mo + PyF101 M-MuLV, PyF101 + Mo M-MuLV induced leukemia with kinetics similar to that of wild-type M-MuLV. Thus, the position of the PyF101 sequences relative to the M-MuLV LTR affected the biological behavior of the molecular construct. Furthermore, PyF101 + Mo M-MuLV induced a different spectrum of neoplastic disease. In comparison with wild-type M-MuLV, which induces a characteristic thymus-derived lymphoblastic lymphoma with extremely high frequency, PyF101 + Mo M-MuLV was capable of inducing both acute myeloid leukemia or thymus-derived lymphoblastic lymphoma, or both. Tumor DNA from both the PyF101 + Mo- and Mo- M-MuLV-inoculated animals contained recombinant proviruses with LTRs that differed from the initially inoculated virus.

Animals↗
PubMed2002

Ancient lineages of non-LTR retrotransposons in the primitive eukaryote, Giardia lamblia.

Mobile elements that use reverse transcriptase to make new copies of themselves are found in all major lineages of eukaryotes. The non-long terminal repeat (non-LTR) retrotransposons have been suggested to be the oldest of these eukaryotic elements. Phylogenetic analysis of non-LTR elements suggests that they have predominantly undergone vertical transmission, as opposed to the frequent horizontal transmissions found for other mobile elements. One prediction of this vertical model of inheritance is that the oldest lineages of eukaryotes should exclusively harbor the oldest lineages of non-LTR retrotransposons. Here we characterize the non-LTR retrotransposons present in one of the most primitive eukaryotes, the diplomonad Giardia lamblia. Two families of elements were detected in the WB isolate of G. lamblia currently being used for the genome sequencing project. These elements are clearly distinct from all other previously described non-LTR lineages. Phylogenetic analysis indicates that these Genie elements (for Giardia early non-LTR insertion element) are among the oldest known lineages of non-LTR elements consistent with strict vertical descent. Genie elements encode a single open reading frame with a carboxyl terminal endonuclease domain. Genie 1 is site specific, as seven to eight copies are present in a single tandem array of a 771-bp repeat near the telomere of one chromosome. The function of this repeat is not known. One additional, highly divergent, element within the Genie 1 lineage is not located in this tandem array but is near a second telomere. Four different telomere addition sites could be identified within or near the Genie elements on each of these chromosomes. The second lineage of non-LTR elements, Genie 2, is composed of about 10 degenerate copies. Genie 2 elements do not appear to be site specific in their insertion. An unusual aspect of Genie 2 is that all copies contain inverted repeats up to 172 bp in length.

Animals↗
PubMed1993

Significant dispersed recurrent DNA sequences in the Escherichia coli genome. Several new groups.

New computer and statistical methods were used to determine significant direct and inverted repeats in the Escherichia coli contig sequence collection of aggregate 1.6 x 10(6) base-pairs. Eight groups of mostly new structural repeat identities were uncovered. Apart from the high statistical significance of these repeat sequences, there are suggestive relationships of the group matches in terms of neighboring genes, of genomic distributions, of their texts, and of their potentials for secondary structure. Four of these groups are relatively numerous, 11 to 26 members, one is in coding sequences and three are in non-coding. The coding group consists of the ATP-activated transmembrane component of a typical high-affinity protein-binding transport system. One of the non-coding groups consists of a special rho-independent transcription termination signal closely following an operon. The gene neighbors of this group often appear to be involved in some way in processing RNA or DNA. A second non-coding group has, for one or both neighboring genes, a component of a system responding to stress or starvation for some nutrient.

Algorithms↗
PubMed1999

Positive co-regulation of the Escherichia coli carnitine pathway cai and fix operons by CRP and the CaiF activator.

Activation of the two divergent Escherichia coli cai and fix operons involved in anaerobic carnitine metabolism is co-dependent on the cyclic AMP receptor protein (CRP) and on CaiF, the specific carnitine-sensitive transcriptional regulator. CaiF was overproduced using a phage T7 system, purified on a heparin column and ran as a 15 kDa protein on SDS-PAGE. DNase I footprinting and interference experiments identified two sites, F1 and F2, with apparently comparable affinities for the binding of CaiF in the cai-fix regulatory region. These sites share a common perfect inverted repeat comprising two 11 bp half-sites separated by 13 bp, and centred at -70 and -127 from the fix transcription start site. They were found to overlap the two low-affinity binding sites, CRP2 and CRP3, determined previously for CRP. Gel shift assays and footprinting experiments suggest that CaiF and CRP bind co-operatively to the F1/CRP2 and F2/CRP3 sites of the intergenic cai-fix region. Moreover, they appeared to serve the simultaneous binding of each other, giving rise to an original multiprotein CRP-CaiF complex enabling RNA polymerase recruitment and local DNA untwisting, at least at the fix promoter. Using random mutagenesis, two CaiF mutants impaired in transcription activation were isolated. The N-terminal A27V mutation affected the structural organization of the activator, whereas the central I62N mutation was suggested to interfere with DNA binding.

Bacterial Proteins↗
PubMed1991

Structure and chromosomal location of the gene for endothelial-leukocyte adhesion molecule 1.

Endothelial-leukocyte adhesion molecule 1 is a cell surface glycoprotein expressed by cytokine-activated endothelium that mediates the adhesion of blood neutrophils. Endothelial-leukocyte adhesion molecule 1 is a member of the selectin family of cell adhesion molecules each of which contain an amino-terminal lectin-like domain, followed by an epidermal growth factor-like domain and a variable number of short consensus repeats similar to those found in complement binding proteins. Genomic clones encoding the ELAM gene were isolated and the organization of the ELAM gene was determined. The gene, which is present in a single copy in the human genome, contains 14 exons spanning about 13 kilobases of DNA. The positions of exon-intron boundaries correlate with the putative functional subdivisions of the protein. Introns are found at similar positions in all of the six complement regulatory repeats, suggesting that these elements arose by internal gene duplication. A consensus TATAA element is located upstream of the transcriptional start site. The ELAM promoter contains an inverted CCAAT box and consensus NF-kappa B- and AP-1-binding sites. The ELAM gene was assigned to the q12 greater than qter region of human chromosome 1 by analysis of human-mouse hybrid cell lines. Two other members of the selectin gene family, the leukocyte adhesion molecule 1 (LAM-1, TQ1, LEC-CAM 1, or Leu-8) and the granule membrane protein 140 (GMP-140, PADGEM, or CD62) have been localized to the long arm of chromosome 1, as have the structurally related complement binding proteins, suggesting that these genes may share a common evolutionary history.

Amino Acid Sequence↗
PubMed1999

SREBP-1 binds to multiple sites and transactivates the human ApoA-II promoter in vitro : SREBP-1 mutants defective in DNA binding or transcriptional activation repress ApoA-II promoter activity.

-Screening of an expression human liver cDNA library resulted in the isolation of several cDNA clones homologous to sterol regulatory element-binding protein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter. DNaseI footprinting of the apoA-II promoter using SREBP-1 (1 to 460) expressed in bacteria identified 5 overall protected regions designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332), AIIHI (-594 to -574), and AIIK (-760 to -743). These regions contain inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities. Transient cotransfection experiments in HepG2 cells showed that SREBP-1 transactivated the -911/29 apoA-II promoter 3.5-fold as well as truncated apoA-II promoter segments that contain 1, 2, 3, or 4 SREBP binding sites. Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB. Despite the strong transactivation of the apoA-II promoter by SREBP-1 we could not demonstrate significant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol and 25-OH-cholesterol. An SREBP-1 mutant lacking the amino-terminal activation domain bound normally to its cognate sites and repressed the apoA-II promoter activity. Repression was also caused by specific amino acid substitutions of Leu, Val, or Gly for Lys359, which affected DNA binding. Repression by the DNA binding-deficient mutants was abolished by deletion of the amino-terminal activation domain (1 to 90) of SREBP-1. Overall, the findings suggest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture. SREBP-1 mutants lacking the activation domain bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II promoter activity, possibly by a squelching mechanism.

Amino Acid Sequence↗
PubMed1998

Purification, cloning, and preliminary characterization of a Spiroplasma citri ribosomal protein with DNA binding capacity.

The rpsB-tsf-x operon of Spiroplasma citri encodes ribosomal protein S2 and elongation factor Ts, two components of the translational apparatus, and an unidentified X protein. A potential DNA-binding site (a 20-base pair (bp) inverted repeat sequence) is located at the 3' end of rpsB. Southwestern analysis of S. citri proteins, with a 30-bp double-stranded oligonucleotide probe (IRS), containing the 20-bp inverted repeat sequence and the genomic flanking sequences, detected an IRS-binding protein of 46 kDa (P46). P46 protein, which displays preferential affinity for the IRS, was purified from S. citri by a combination of affinity and gel filtration chromatographies. The native form of P46 seems to be homomultimeric as estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration. A 3.5-kilobase pair S. citri DNA fragment comprising the P46 gene and flanking sequences was cloned and sequenced. Sequence analysis of this DNA fragment indicated that the P46 gene is located within the S10-spc operon of S. citri at the position of the gene coding for ribosomal protein L29 in the known S10-spc operons. The similarity between the N-terminal domain of P46 and the L29 ribosomal protein family and the presence of a 46-kDa IRS-binding protein in S. citri ribosomes indicated that P46 is the L29 ribosomal protein of S. citri. We suggest that P46 is a bifunctional protein with an L29 N-terminal domain and a C-terminal domain involved in IRS binding.

Bacterial Outer Membrane Proteins↗
PubMed1999

Dual control by regulatory gene fdsR of the fds operon encoding the NAD+-linked formate dehydrogenase of Ralstonia eutropha.

The transcriptional regulator gene fdsR was identified 150 bp upstream of the divergently oriented fdsGBACD operon encoding the soluble, NAD+-linked formate dehydrogenase in the chemoautotrophic bacterium Ralstonia eutropha H16. Its deduced product, FdsR, displays a basal sequence similarity to the regulatory proteins of the LysR family. The carboxy-terminal domain of FdsR contains a short region that is conserved in formate dehydrogenases. Deletion of fdsR revealed a dual regulatory effect of FdsR on the fds operon by acting as transcriptional activator in the presence of formate or as repressor in the absence of formate. Studies with fdsR transcriptional fusions also suggested a negative autoregulation of the gene. A promoter structure resembling sigma70-dependent promoters from Escherichia coli was identified upstream of the fdsR transcriptional start site. FdsR purified to homogeneity after overexpression of fdsR in E. coli is a 130 kDa homotetramer binding to the fds control region located between the fdsR and fdsG genes. Formate significantly increased the binding affinity of FdsR for this region. Two FdsR binding sites characterized by the inverted-repeat structure ATANG-N10-CNTAT were identified. The regulatory pattern found in R. eutropha was also observed in the heterologous host E. coli and results from a novel mode of control of formate dehydrogenase genes.

Amino Acid Sequence↗
PubMed2002

FOR, a novel orphan nuclear receptor related to farnesoid X receptor.

We have identified and characterized a new amphibian orphan member of the nuclear receptor superfamily and termed it FOR1 (farnesoid X receptor (FXR)-like Orphan Receptor) because it shares the highest amino acid identity with the mammalian FXR. We also identified a variant of FOR1, called FOR2, which has 15 additional C-terminal amino acids. Both variants include an unusual insertion of 33 amino acids in the helix 7 region of the canonical ligand binding domain sequence, suggesting a unique structure for FOR. Northern blot analysis demonstrates that the FOR gene is highly expressed in adult and tadpole liver, kidney, and tail bud stage of the embryo. Detailed expression analysis using in situ hybridization indicates that FOR expression is first detectable at stage 30/31 in the presumptive liver region lasting until stage 41 with a peak level evident at stage 35/36. FOR forms heterodimeric complexes with retinoid X receptor (RXR) as demonstrated by biochemical and mammalian two-hybrid approaches. Gel mobility shift assays demonstrate that FORs form specific DNA-protein complexes on an FXR binding element consisting of an inverted repeat DNA element with 1 nucleotide spacing (IR1) from the phospholipid transfer protein gene promoter. Finally, although FORs do not exhibit constitutive transcriptional activity, frog gallbladder extract significantly augments the transcriptional activities of FORs.

Amino Acid Sequence↗
PubMed1977

Structure of the inverted terminal repetition of adenovirus type 2 DNA.

Several secondary structure features involving the ends of single strands of adenovirus type 2 DNA have been studied by electron microscopy by both the gene 32-ethidium bromide technique and a modification of the standard formamide-cytochrome c technique. A duplex stem of length 115 +/- 10 nucleotide pairs due to pairing between the two members of the inverted terminal repetition is observed in the single-stranded circles that form upon annealing single-stranded linear molecules. This duplex stem is shown to lie at the ends of the DNA by using several reference markers: (i) a newly discovered secondary structure feature (a loop of length ca. 500 nucleotides with a 20-nucleotide pair duplex stem) that maps 73% of the full length from the left end of the molecule and (ii) a duplex region due to a hybridized restriction fragment. There is also some secondary structure within each end of linear single strands. There is some variation in the morphology of the end strucures, and we propose that these involve base pairing, as in a tRNA clover leaf, rather than an exact single hairpin-type inverted repeat. These observations are consistent with the hypothesis that there is a foldback structure at the 3' ends of the DNA that functions as a primer for the initiation of replication.

Adenoviruses, Human↗
PubMed1997

Identification of transposon-like elements in non-coding regions of tomato ACC oxidase genes.

1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyses the terminal step in ethylene biosynthesis, is encoded by a small multigene family in tomato that is differentially expressed in response to developmental and environmental cues. In this study we report the isolation and sequencing of approximately 2 kb of 5'-flanking sequence of three tomato ACC oxidase genes (LEACO1, LEACO2, LEACO3) and the occurrence of class I and class II mobile element-like insertions in promoter and intron regions of two of them. The LEA CO1 upstream region contains a 420-bp direct repeat which is present in multiple copies in the tomato genome and is very similar to sequences in the promoters of the tomato E4 and 2A11 genes. The region covering the repeats resembles the remnant of a retrotransposon. Two copies of a small transposable element, belonging to the Stowaway inverted repeat element family, have been found in the 5'-flanking sequence and the third intron of LEACO3.

Amino Acid Oxidoreductases↗
PubMed1988

An enhancer sequence instability that diversifies the cell repertoire for expression of a murine leukemia virus.

Studies of recombinants between murine leukemia viruses (MuLVs) that cause thymic or erythroid leukemias have shown that enhancer sequences in the long-terminal repeats (LTRs) can determine the target tissues for pathogenesis. It has been inferred that the enhancers may specifically target viral expression into the cells that then become neoplastic. However, the neoplasms in those studies formed after latencies and contained ultimate viruses (called MCFs) that differed from the injected viruses in their enhancer sequences and envelope (env) genes. Transcriptional activities of LTRs from these proximal and ultimate viruses have not been thoroughly analyzed in different hematopoietic lineages. We present evidence that the enhancer of Friend spleen focus-forming virus (SFFV), an ultimate erythroleukemogenic retrovirus, contains an unstable 42-nucleotide direct repeat. Other ultimate erythroleukemogenic MuLVs (Friend MCFs) contain an enhancer nearly identical to that of SFFV both in its sequence and in its specific instability. The instability occurs in sequences that contain inverted repeats and we propose that it occurs by a simple reverse transcriptase hop mechanism. We constructed plasmids that contain the two forms of the SFFV LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, and we compared these in transient transfection assays with LTR-CAT plasmids constructed from Friend and Moloney MuLVs. The assays employed erythroleukemia cells, thymic lymphoma cells, and fibroblasts. The tropisms of expression correlated only weakly with tissue specificities of pathogenesis and each LTR was active in all cells. The SFFV 42-nucleotide duplication reduced expression in erythroid cells and increased expression in fibroblasts. We conclude that retroviral enhancers do not stringently direct gene expression into specific cell lineages, but on the contrary they are leaky and contain replicative instabilities that also may facilitate viral entrenchment throughout the host. These results have important implications for understanding murine retroviral evolution and the multi-step process of leukemogenesis.

Base Sequence↗
PubMed1998

Genetic profile of pNOB8 from Sulfolobus: the first conjugative plasmid from an archaeon.

The complete nucleotide sequence of the archaeal conjugative plasmid, pNOB8, from the Sulfolobus isolate NOB8-H2, was determined. The plasmid is 41,229 bp in size and contains about 50 ORFs. Several direct sequence repeats are present, the largest of which is a perfect 85-bp repeat and a site of intraplasmid recombination in foreign Sulfolobus hosts. This recombination event produces a major deletion variant, pNOB8-33, which is not stably maintained. Less than 20% of the ORFs could be assigned putative functions after extensive database searches. Tandem ORFs 315 and 470, within the deleted 8-kb region, show significant sequence similarity to the protein superfamilies of ParA (whole protein) and ParB (N-terminal half), respectively, that are important for plasmid and chromosome partitioning in bacteria. A putative cis-acting element is also present that exhibits six 24-mer repeats containing palindromic sequences which are separated by 39 or 42 bp. By analogy with bacterial systems, this element may confer plasmid incompatibility and define a group of incompatible plasmids in Archaea. Although several ORFs can form putative trans-membrane or membrane-binding segments, only two ORFs show significant sequence similarity to bacterial conjugative proteins. ORF630b aligns with the TrbE protein superfamily, which contributes to mating pair formation in Bacteria, while ORF1025 aligns with the TraG protein superfamily. We infer that the conjugative mechanism for Sulfolobus differs considerably from known bacterial mechanisms. Finally, two transposases were detected; ORF413 is flanked by an imperfect 32-bp inverted repeat with a 5-bp direct repeat at the ends, and ORF406 is very similar in sequence to an insertion element identified in the Sulfolobus solfataricus P2 genome.

Amino Acid Sequence↗
PubMed1992

Cloning and sequencing of the genes encoding glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase (gap operon) from mesophilic Bacillus megaterium: comparison with corresponding sequences from thermophilic Bacillus stearothermophilus.

The structural genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and the N-terminal part of triosephosphate isomerase (TIM) from mesophilic Bacillus megaterium DSM319 have been cloned as a gene cluster (gap operon) by complementation of an Escherichia coli gap amber mutant. Subsequently, the entire tpi gene, encoding TIM, was isolated by colony hybridization using a homologous probe. Nucleotide (nt) sequence analysis revealed an unidentified open reading frame (urf1) of 1029 bp located 50 nt upstream from the start codon of the gap gene. Gene expression from subclones containing different coding regions was studied by enzyme assay and SDS-PAGE. Both GAPDH and TIM are synthesized in transformed E. coli cells, whereas PGK is not. There is no unequivocal evidence for urf1 expression. Two putative promoter sites are present: one 100 nt upstream from urf1 and one 200 nt upstream from the pgk gene. An inverted repeat following the second promoter site is postulated to be involved in the transcriptional regulation of the operon. Each coding region shows a G+C content of 40% attained by the adaptation of the G+C content of the third base in the codon to compensate the G+C content of the first and second bases. The deduced amino acid (aa) sequences of B. megaterium GAPDH, PGK and TIM were compared with those from the thermophilic Bacillus stearothermophilus by antisymmetrical matrices. The detected characteristic thermophilic-mesophilic exchange pattern concerning aa substitutions between hydrophobic-polar and charged-charged residues corresponds to data obtained for thermophilic and mesophilic lactate dehydrogenases (LDH). The determination of the thermostability of these enzymes revealed two regions of stability for B. megaterium TIM at high enzyme concentrations. Heat treatment seems to be responsible for the conversion of two differently active conformations or the induction of a new quaternary structure.

Amino Acid Sequence↗
PubMed1985

The partition functions of P1, P7, and F miniplasmids.

The partition regions of P1, P7, and F miniplasmids are discrete DNA sequences of about 3 kb in length that will promote accurate partition of hybrid plasmids independent of the source of replication functions or the position or orientation of the elements. Each of the par regions seems to be very similarly organized, with open reading frames for essential proteins and a terminal site which appears to be analogous to the centromere of eukaryotic cells. When cloned, these terminal sites exert incompatibility against their respective parent plasmids presumably because they can compete with the parent plasmids as substrates for partition. We have determined the complete DNA sequence of the P1 par region. In addition to the open reading frame for the essential parA protein (42-44 kd), the region contains a second open reading frame which could encode a 38-kd protein. The 2 large open reading frames appear to form an operon that is negatively regulated from a site adjacent to the promoter and responds to the par gene products in trans. Both this site and the downstream "centromere" site, incB, contain blocks of extremely AT-rich sequences, which are postulated to be binding sites for par proteins. The incB and upstream AT-rich regions both contain 20-bp imperfect inverted repeats. Further downstream from the minimal incB sequence (172 bp) lies an additional region which is essential for partition. The further analysis of the P1 par region should be greatly facilitated by the finding that it can function in cis to stabilize pBR322 vectors under conditions where the copy number of pBR322 is reduced.

Amino Acid Sequence↗