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PubMed1996

Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame.

The construction of a full-length clone of the avian coronavirus infectious bronchitis virus (IBV) defective RNA (D-RNA), CD-91 (9,080 nucleotides [Z. Penzes et al., Virology 203:286-293]), downstream of the bacteriophage T7 promoter is described. Electroporation of in vitro T7-transcribed CD-91 RNA into IBV helper virus-infected primary chick kidney cells resulted in the production of CD-91 RNA as a replicating D-RNA in subsequent passages. Three CD-91 deletion mutants were constructed--CD-44, CD-58, and CD-61--in which 4,639, 3,236, and 2,953 nucleotides, respectively, were removed from CD-91, resulting in the truncation of the CD-91 long open reading frame (ORF) from 6,465 to 1,311, 1,263, or 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively. Electroporation of in vitro T7-transcribed RNA from the three constructs into IBV helper virus-infected cells resulted in the replication and packaging of CD-58 and CD-61 but not CD-44 RNA. The ORF of CD-61 was further truncated by the insertion of stop codons into the CD-61 sequence by PCR mutagenesis, resulting in constructs CD-61T11 (ORF: nucleotides 996 to 1,058, encoding 20 amino acids), CD-61T22 (ORF: nucleotides 996 to 2,294, encoding 432 amino acids), and CD-61T24 (ORF: nucleotides 996 to 2,450, encoding 484 amino acids), all of which were replicated and packaged to the same levels as observed for either CD-61 or CD-91. Analysis of the D-RNAs showed that the CD-91- or CD-61-specific long ORFs had not been restored. Our data indicate that IBV D-RNAs based on the natural D-RNA, CD-91, do not require a long ORF for efficient replication. In addition, a 1.4-kb sequence, corresponding to IBV sequence at the 5' end of the 1b gene, may be involved in the packaging of IBV D-RNAs or form part of a cis-acting replication element.

Animals↗
PubMed1990

The E2 trans-activator can act as a repressor by interfering with a cellular transcription factor.

The E2 open reading frame (ORF) of the bovine papillomavirus (BPV-1) encodes a family of site-specific DNA-binding proteins. The full-length protein is a transcriptional activator, whereas the polypeptides that contain only the carboxy-terminal domain are repressors. Here we show that the trans-activator can work as a repressor of transcription for one of the BPV-1 promoters by binding to a DNA sequence required for basal activity of the promoter. This operator site is defined as a 12-bp sequence that lies immediately downstream of the cap site. The operator DNA contains sequences that are defined genetically and biochemically as being important for basal level promoter activity. Furthermore, this site has been shown to be protected in a DNase footprint assay using fractionated HeLa cell extracts. The repression does not simply result from E2 blocking RNA polymerase initiation or elongation, because a strong E2-binding site placed at the operator has no repressive effect on transcription when the basal target sequence is placed independently upstream of the promoter. Thus, this is an interesting parallel to a theme well known in prokaryotes, where some site-specific DNA-binding proteins can work as either activators or repressors. In this system, as well as in the prokaryotic systems, the precise position of the binding site relative to other cis signals at the promoter determines the nature of the effects.

Base Sequence↗
PubMed1985

A new putative gene in the mitochondrial genome of Saccharomyces cerevisiae.

The 2200-bp ori2-ori7 region of the mitochondrial (mt) genome of Saccharomyces cerevisiae has been sequenced on the genome of a petite, b7, excised at those ori sequences from wild-type strain B. The region contains an open reading frame, ORF5, which is transcribed into a 900-nucleotide (nt) RNA in both the parental wild-type strain and its derived petite, b7. This RNA uses as a template the strand used by most mt transcripts. Its start point is located 337 nt upstream of ORF5; and a messenger termination site has been found 900 nt downstream of the initiation site. These data suggest that ORF5 is a new mitochondrial gene. The G + C content of ORF5 is only 15.7%; 90% of the G + C base pairs of ORF5 are comprised in a palindromic G + C cluster similar to that present in the varl gene. The coding capacity of ORF5 is 46 amino acids (aa), mainly represented by methionine, phenylalanine, arginine, valine, asparagine, isoleucine and tyrosine. The aa composition and the codon usage of ORF5 are reminiscent of those of varl and of other intergenic ORFs.

Amino Acid Sequence↗
PubMed1996

Flagellar genes from Rhodobacter sphaeroides are homologous to genes of the fliF operon of Salmonella typhimurium and to the type-III secretion system.

A flagellar region of the genome of Rhodobacter sphaeroides was cloned and sequenced. Three ORFs were identified and arranged in the same order as fliH, fliI and fliJ of Salmonella typhimurium (St). ORF2 is highly similar to FliI from St (49% similarity) showing Walker's A and B motifs. Similar scores were found with proteins of the type-III secretion system of virulence factors. ORF3 shows 16.4 and 11.1% similarity to FliJ from St and Bacillus subtilis, respectively. This work also shows that ORF3 is similar to HrpJ5 from Pseudomonas syringae (19.2% similarity). It was found that ORF2 and ORF3 start immediately downstream from the adjacent coding region, suggesting a single transcriptional unit.

Amino Acid Sequence↗
PubMed1993

Identification of a cis acting element responsible for muscle specific expression of the c-mos protooncogene.

A series of deletion constructs of the 5' flanking region of rat c-mos gene was positioned upstream to the CAT gene and transfected into muscle and non-muscle cells. CAT activities revealed that a region located downstream of a TATA box and containing the proximal transcription start site is the muscle c-mos promoter. This promoter is more efficient in L6 alpha 1 myoblasts than in L6 alpha 1 myotubes but not in C3H10T1/2 cells. Gel shift assays demonstrated that nuclear proteins from myoblasts and myotubes formed complexes migrating differently. Footprinting analyses showed that nuclear proteins from L6 alpha 1 myoblasts protected a DNA fragment located at position nt -979 to nt -938 relative to the first ATG of the rat c-mos ORF while nuclear proteins from myotubes protected the DNA between nt -998 to nt -928. Furthermore one of protein - DNA complexes containing the proximal transcription start site, included a consensus sequence TGTC(AGT/TCG)CC(A/T)G present in the initiator element (Inr) of several genes. Southwestern blot analysis pointed to a 82kDa polypeptide as a potential candidate for trans acting factor in myoblasts. In L6 alpha 1 myotubes this polypeptide is replaced by other proteins of 40-42kDa and 82kDa. An interplay between these two complexes may constitute a developmental as well as a physiologically regulated mechanism that modulates c-mos expression during the early stages of myogenesis.

Animals↗
PubMed1992

The region essential for efficient autonomous replication of pSa in Escherichia coli.

Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.

Amino Acid Sequence↗
PubMed2003

Phylogenetic reconstruction of Gram-positive organisms based on comparative sequence analysis of molecular chaperones from the ruminal microorganism Ruminococcus flavefaciens FD-1.

Primers designed on the basis of nucleotide sequences conserved in DnaK and GroEL from Gram-positive organisms were used to PCR amplify internal regions of the cognate genes from the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1. Genome walking was then utilized to elucidate the remainder of the sequences in addition to upstream and downstream regions. The full sequence of the gene encoding the GroES protein (groES) was found directly upstream from groEL. The deduced amino acid sequence of the groEL gene showed the highest homology with the amino acid sequence of the Clostridium thermocellum GroEL protein (72% amino acid identity). Similarly, translation of the groES nucleotide sequence showed highest homology to the C. thermocellum GroES protein (61% amino acid identity). Analysis of the upstream region of this chaperonin operon revealed a CIRCE regulatory element 45 bp upstream from the putative start of the groES ORF. The deduced amino acid sequence of the putative dnaK gene showed the highest homology with the amino acid sequence of the Clostridium acetobutylicum DnaK protein (68% amino acid identity). Phylogenetic analyses based on the translated sequences reiterate this relationship between R. flavefaciens and the Clostridia. However, when the nucleotide sequences of Gram-positive organisms are analyzed, a different topology occurs of the relationship between high- and low-G+C Gram-positive organisms to the 16S rRNA interpretation.

Animals↗
PubMedDate not supplied

Characterization of the hrpJ and hrpU operons of Pseudomonas syringae pv. syringae Pss61: similarity with components of enteric bacteria involved in flagellar biogenesis and demonstration of their role in HarpinPss secretion.

The hrp/hrmA gene cluster of Pseudomonas syringae pv. syringae Pss61 has been shown to form a minimum genetic unit sufficient to enable nonpathogenic bacteria, such as Escherichia coli, to elicit the hypersensitive response associated with disease resistance. The biochemical functions of most of these genes have not been established. The nucleotide sequence of a 4.3-kb SstI-BglII fragment carrying hrp apparent translational units V, VI, and VII revealed one partial open reading frame (ORF) and five complete ORFs producing 35,126-, 48,866-, 17,308-, 20,482-, and 26,364-Da gene products (hrpJ3, J4, J5, U1, U2, respectively). The production of these proteins was confirmed by using T7 RNA polymerase-directed expression. The partial ORF was found to be identical to the C terminus of HrpJ2. The absence of apparent transcriptional terminators and promoters between hrpI (hrpJ2), hrpJ3, hrpJ4, and hrpJ5 together with the observation that the HrpL-dependent hrpJ promoter directs expression of hrpJ3-J5 indicates that these genes form a single operon controlled by the HrpL-dependent hrpJ promoter. A second HrpL-dependent promoter consensus sequence was also identified upstream of hrpU1 and demonstrated to function as a HrpL-dependent promoter, thus indicating that hrpU1, hrpU2, and additional downstream genes may be part of a second operon. The deduced product of hrpJ3 exhibits similarity to FliG of Salmonella typhimurium, a cytoplasmic protein that regulates flagellar rotation and biogenesis. HrpJ4 shares extensive similarity with the FliI family of ATPase-like proteins and retains the known functional domains conserved among this family of proteins. HrpJ5 has properties similar to the S. typhimurium FliJ. Neither HrpU1 nor HrpU2 exhibit significant similarity to known proteins. Secretion of HarpinPss by E. coli MC4100 transformants carrying pHIR11::TnphoA derivatives was blocked in hrpJ4, J5, and U2 mutants. In view of the previously reported similarity of HrpJ2 to the LcrD super-family that includes FlhA, these results predict that the gene products of the hrpJ and hrpU operons form an inner membrane complex for translocation of proteins similar to that used by the flagellar biogenesis system of S. typhimurium.

Amino Acid Sequence↗
PubMed1997

Reduced antigen production by hepatitis B virus harbouring nucleotide deletions in the overlapping X gene and precore-core promoter.

Hepatitis B virus (HBV) genomes with deletions in the precore-core (preC-C) promoter have been detected in HBV infections without serological markers. To address whether the mutations are responsible for the reduced production of virus antigenes, either an 8 bp (8d, position 1763 to 1770) or a 20 bp (20d, 1753 to 1772) deletion was created in a wild-type (wt) HBV clone. Both mutations cause premature termination of the overlapping X ORF. When introduced into HepG2 cells, both mutants produced reduced amounts of HBsAg, HBcAg and HBeAg, but released the same or more virion-associated DNA compared with the wt. A co-transfection of the 20d mutant with a small amount of intact X gene resulted in a 3-fold increase of HBcAg production compared to transfection with either the 20d or wt alone. When the promoter region was cloned into CAT plasmids, the 8d preC promoter showed weak activity and its initiation site was shifted 6 to 10 bp downstream. The preC promoter activity of 20d was not detectable by CAT ELISA and 5' RACE. The levels of C transcripts of both mutants were higher than that of the wt, and their start sites were not altered. Therefore, the deletions cause the reduction of HBsAg, HBcAg and HBeAg although the mutant viruses can still replicate in cultured cells. The reduction of HBeAg is due to both the reduced preC promoter activity and the defect in HBx. The reduction of HBcAg is due to the disrupted X gene, despite augmented C promoter activity.

Amino Acid Sequence↗
PubMed2001

Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site.

We have previously examined the transcription and splicing of open reading frames (ORFS) 71 (K13), 72, and 73 of Kaposi's sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and latency-associated nuclear antigen [LANA]) are transcribed from a common transcription start site in BCP-1 cells during both latency and the lytic cycles. The resulting transcript is spliced to yield a 5.32-kb message encoding LANA, vCyclin, and vFLIP and a 1.7-kb bicistronic message encoding vCyclin and vFLIP. To investigate whether the vFLIP protein could be expressed from this vCyclin/vFLIP message, we utilized a bicistronic luciferase reporter system. The genes for Renilla and firefly luciferases (which utilize different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of vFLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative internal ribosome entry site (IRES) sequences and the other by cap-dependent ribosome scanning, was used to compare the activities of the different DNA fragments. A minimum fragment of 233 bp within the coding region of vCyclin was found to direct efficient expression of the downstream cistron (firefly luciferase). The activity of this IRES was orientation dependent and unaffected by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism through which KSHV controls protein expression.

Base Sequence↗
PubMed2006

A two-step method for the introduction of single or multiple defined point mutations into the genome of Saccharomyces cerevisiae.

The introduction of defined mutations into open reading frames (ORF) or non-translated regions of the genome is important to study of the structure-function relationship of amino acid residues in proteins or that of sequence motifs at the genome level. We describe a simple two-step method for the introduction of defined single or multiple point mutations into the genome of Saccharomyces cerevisiae. This method circumvents the need for plasmid-based mutagenesis and thus ensures homogenous expression of the gene of interest within the cell population. It is based on the introduction of a selectable marker downstream of the gene of interest. This marker is then amplified with a gene-specific primer that harbours the desired point mutation, creating a selectable marker-tagged mutant version of the gene of interest. The mutant fragment is then integrated into the genome of a wild-type strain through homologous recombination. Successive rounds of amplification of the mutant loci with primers that introduce additional point mutations upstream of existing mutations will generate multiple defined mutations within a single gene. As a proof of principle, we have employed this method to generate a temperature-sensitive mutant version of the plasma membrane ATPase, pma1-7, which bears two point mutations (Pro434Ala and Gly789Ser). Phenotypic analysis of a pma1-7 haploid strain indicates that this allele has the same characteristics as the original pma1-7 allele. It confers a temperature-sensitive growth phenotype and the newly synthesized Pma1-7 protein is unstable and rapidly degraded.

Alleles↗
PubMed1994

Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F.

Previous studies have shown that the capsular polysaccharide synthesis (cps) locus of the type 19F Streptococcus pneumoniae strain SSZ was closely linked to a copy of the insertion sequence IS1202 (J.K. Morona, A. Guidolin, R. Morona, D. Hansman, and J.C. Paton, J. Bacteriol. 176:4437-4443, 1994). In the present study, we used plasmid insertion and rescue and inverse PCR to clone 6,322 bp of flanking DNA upstream of IS1202. Sequence analysis indicated that this region contains six complete open reading frames (ORFs) and one partial ORF that are arranged as a single transcriptional unit. Chromosomal disruption of any of these ORFs in a smooth-type 19F strain leads to a rough (unencapsulated) phenotype, indicating that this operon is essential for capsule production. The ORFs have therefore been designated cps19fA to cps19fG, where cps19fA is the first gene of the type 19F cps locus. Furthermore, many of the gene products from this incomplete operon exhibit strong similarities to proteins known to be involved in the production of capsular polysaccharide, exopolysaccharide, teichoic acid, enterobacterial common antigen, and lipopolysaccharide from numerous other bacterial species. This has allowed us to propose functions for many of the type 19F cps gene products. Southern hybridization studies reveal that cps19fA and cps19fB are conserved among all 12 pneumococcal serotypes tested, whereas genes downstream of cps19fB are conserved among some, but not all, of the serotypes tested.

Amino Acid Sequence↗
PubMed1997

The Staphylococcus aureus collagen adhesin-encoding gene (cna) is within a discrete genetic element.

Although the gene (cna) encoding the Staphylococcus aureus (Sa) collagen adhesin is not present in all strains, the DNA both upstream and downstream of cna is present in all Sa strains. Using oligo primers corresponding to the conserved nt flanking cna and template DNA from Sa strains that do not encode cna, we amplified a 372-bp fragment. These results illustrate that the conserved regions upstream and downstream of cna are contiguous in strains that do not encode cna. Using primers corresponding to the conserved flanking DNA together with primers corresponding to the 5' and 3' ends of cna, we also amplified DNA fragments containing the junctions between the cna genetic element and the conserved flanking sequences. Sequence comparisons of the amplification products from four cna negative and four cna positive strains revealed that cna is within a discrete genetic element that extends 202 bp upstream from the cna start codon and 100 bp downstream of the cna stop codon. Sequence analysis of the ends of the cna element did not reveal any of the repeats characteristic of transposable elements. These results suggest that cna may be part of a larger element (e.g., a phage) that may or may not contain cna. Alternatively, cna may be a subject to a precise excision event resulting in its deletion from the chromosome. Based on sequence analysis of the flanking DNA amplified from strains that do not encode cna, the presence of a cna genetic element does not disrupt an ORF.

Adhesins, Bacterial↗
PubMed1994

Cloning and expression of genes from the genomic region between genes cd and 30 of bacteriophage T4.

Ten open reading frames (ORFs) from the cd-30 region of bacteriophage T4, as well as genes 31 and rIII, have been cloned, and all were found to be expressed in a T7 RNA polymerase system. Expression data confirm the existence of genes 31.2, 31.1, 31, rIII, 30.9, 30.8, 30.7, 30.6, 30.5, 30.4, 30.3 and 30.2, which predict peptides of 78, 102, 111, 82, 58, 110, 121, 95, 65, 68, 152 and 278 amino acids (aa), respectively. The major product of gene 30.9 is a basic peptide initiated at the second AUG codon that is found 42 nucleotides downstream and in frame from the first AUG. A plasmid carrying gene 30.3 expresses two different peptides, one of which is of the size predicted for gp30.3. By site-directed mutagenesis we have shown that the smaller peptide is encoded by gene 30.3' which is completely embedded within gene 30.3, but in a different reading frame. Gene 30.3' encodes a 75-aa basic peptide with its C terminus rich in charged aa.

Amino Acid Sequence↗
PubMed1997

A novel delta-endotoxin gene cryIM from Bacillus thuringiensis ssp. wuhanensis.

A new cryI-related sequence designated cryIM was cloned using an immunoscreening technique from ssp. wuhanensis of Bacillus thuringiensis (BT), previously reported to produce multiple Cry proteins [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034]. Analysis of the cryIM nucleotide sequence revealed an ORF, BTII-type promoter-like sequence, peculiar for such genes, a translation initiation element and a putative transcription terminator. Nevertheless, its product was not previously found in the crystals of the host strain [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034] which shows its weak or absent natural expression. The amino acid sequence of 1151 residues encoded by the continuous reading frame of cryIM is not identical but is essentially similar to the other delta-endotoxins of the CryI class. An IS231-like sequence was found 400 bp downstream of the cryIM stop codon and a fragment of the cryIAb gene was located 3 kb upstream of its initiator codon in the same orientation. Artificial expression of the cloned gene in E. coli under the control of the lacZ promoter allowed us to obtain its hypothetical protein product.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and analysis of transcription initiation in the Anagrapha falcifera multiple nucleocapsid polyhedrosis virus polyhedrin gene.

Using Autographa californica multiple nucleocapsid polyhedrosis virus (AcMNPV) polyhedrin to probe the Southern blots of Anagrapha falcifera multiple nucleocapsid polyedrosis virus (AfMNPV), we identified the location of the AfMNPV polyhedrin gene within the 7.2 kb EcoRI fragment. The 7.2 kb EcoRI fragment of AfMNPV was cloned and the nucleotide sequences of the polyhedrin coding region and its flanking regions were determined. Nucleotide sequence analysis indicated the presence of an open reading frame (ORF) of 735 nucleotides (nt) which could encode 245 amino acids with a predicted molecular mass of 29 kDa. The nucleotide sequences within the coding region of the AfMNPV polyhedrin shared 80% similarity with the polyhedrin gene from AcMNPV but were most closely related to Bombyx mori NPV with 92% sequence identity. The size of the AfMNPV polyhedrin mRNA, determined by the Northern blot analysis, was estimated to be 1200 nt. The consensus promoter sequence (ATAAG) for the baculovirus very late gene was also observed. Two degenerate poly(A) tailing signals were found immediately downstream of the translational stop codon. The transcription initiation site, mapped by primer extension analysis, was found to be at T located 24 nt upstream from the A of the translation initiation codon. This site is located 26 nt downstream from the second A of the consensus TAAG, the transcription initiation site of most other NPVs.

Amino Acid Sequence↗
PubMed1997

The 5'-terminal 32 basepairs conserved between genome segments A and B contain a major promoter element of infectious bursal disease virus.

The regions of the infectious bursal disease virus (IBDV) genome with regulatory function are not known. In the present study, progressively deleted lengths of the 5' noncoding region of segment A were constructed in pGL3 vectors having SV40 enhancer or promoter, and a luciferase (LUC) reporter gene. Transient transfections of the constructs made in a promoter-less pGL3-Enhancer vector when transfected in Vero cells and the lysates assayed for LUC expression, allowed the localization of maximal activity to the 32-nucleotide stretch (precursor polyprotein ORF positions -131 to -100), which is highly conserved at the 5' end of both genome segments. This fragment, when evaluated in parallel in an enhancer-less pGL3-Promoter vector demonstrated no activity. To determine if this region is recognized by IBDV replicative proteins, we engineered modifications in an enhancer-less pGL3-Promoter vector where the terminal 32-bp fragment, the full-length noncoding region, or the noncoding region with the 32-bp fragment deleted was positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the LUC gene. Transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in the endogenous generation of recombinant viral-LUC RNAs containing the 5' terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the infected cell lysates showed up-regulated expression of LUC only with constructs containing the 32-bp fragment in the minus-sense orientation. Deletion of this 32-bp fragment abolished such LUC expression. We therefore conclude that the 5'-terminal 32 base pairs of genomic segment A contain a major promoter element in IBDV. In addition, our results show that IBDV replicative proteins recognize and transcribe single-stranded RNA in vivo.

Animals↗
PubMed1995

A gene from the hyperthermophile Pyrococcus furiosus whose deduced product is homologous to members of the prolyl oligopeptidase family of proteases.

The mlr-2 gene from the hyperthermophilic archaeum Pyrococcus furiosus was identified from a family of clones whose expression was influenced by the presence of maltose in the medium. The sequence of 2100 bp of DNA containing mlr-2 and its flanking regions revealed a 616-amino-acid (71 kDa) open reading frame (ORF). The ORF's initiation codon appeared 10 nt into the mlr-2 message and was not preceded by any apparent ribosome-binding site. The deduced product shared homology with prolyl endopeptidases from both eukaryotic and eubacterial sources (52-57% similarity, 30-37% identity) and signature domains containing the Ser-Asp-His triad, which is characteristic of this family of proteases, were present. Northern blot experiments revealed the presence of an approx. 2.0-kb transcript in P. furiosus extracts, corresponding in length to that expected from mlr-2 expression. Initiation of transcription occurred 23 bp downstream from a putative BoxA promoter element.

Amino Acid Sequence↗