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PubMed2000

The ycf 9 (orf 62) gene in the plant chloroplast genome encodes a hydrophobic protein of stromal thylakoid membranes.

There are still some open reading frames, orfs, with unknown function in the higher plant chloroplast genome. Of these conserved orfs, designated as ycfs (hypothetical chloroplast open reading frames), one is ycf 9 (orf 62) in the transcription unit with the psbC and psbD genes. The aim of this work was to investigate the function of ycf 9 by insertional inactivation of the gene with a selectable marker cassette, consisting of the aadA coding region connected to the trc promoter and rrnB terminator. This cassette was inserted 19 bp downstream from the start of the coding region of the tobacco ycf 9 gene. Two DNA constructs with the aadA cassette in opposite orientations were precipitated on 1 micron gold particles and delivered into leaves of Nicotiana tabacum, cultivar Samsun, by the biolistic method. Spectinomycin-resistant plants regenerated following bombardment with only the construct containing the aadA gene in the opposite orientation as ycf 9. In spite of several subsequent regeneration cycles on spectinomycin, the transplastomic plants did not reach homoplasmicity. This suggests that the ycf 9 gene product is essential for chloroplast function. Using a polyclonal antibody raised against the inner part of the gene product, the polypeptide was localized in the stromal thylakoid membranes of chloroplasts.

Amino Acid Sequence↗
PubMed2006

SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes.

Expression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3'-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins.

Amino Acid Sequence↗
PubMed1993

Rhs elements of Escherichia coli K-12: complex composites of shared and unique components that have different evolutionary histories.

The complete sequences of the RhsB and RhsC elements of Escherichia coli K-12 have been determined. These sequence data reveal a new repeated sequence, called H-rpt (Hinc repeat), which is distinct from the Rhs core repetition that is found in all five Rhs elements. H-rpt is found in RhsB, RhsC, and RhsE. Characterization of H-rpt supports the view that the Rhs elements are composite structures assembled from components with very different evolutionary histories and that their incorporation into the E. coli genome is relatively recent. In each case, H-rpt is found downstream from the Rhs core and is separated from the core by a segment of DNA that is unique to the individual element. The H-rpt's of RhsB and RhsE are very similar, diverging by only 2.1%. They are 1,291 bp in length, and each contains an 1,134-bp open reading frame (ORF). RhsC has three tandem copies of H-rpt, all of which appear defective in that they are large deletions and/or have the reading frame interrupted. Features of H-rpt are analogous to features typical of insertion sequences; however, no associated transposition activity has been detected. A 291-bp fragment of H-rpt is found near min 5 of the E. coli K-12 map and is not associated with any Rhs core homology. The complete core sequences of RhsB and RhsC have been compared with that of RhsA. As anticipated, the three core sequences are closely related, all having identical lengths of 3,714 bp each. Like RhsA, the RhsB and RhsC cores constitute single ORFs that begin with the first core base. In each case, the core ORF extends beyond the core into the unique sequence. Of the three cores, RhsB and RhsA are the most similar, showing only 0.9% sequence divergence, while RhsB and RhsC are the least similar, diverging by 2.9%. All three cores conserve the 28 repetitions of a peptide motif noted originally for RhsA. A secondary structure is proposed for this motif, and the possibility of its having an extracellular binding function is discussed. RhsB contains one additional unique ORF, and RhsC contains two additional unique ORFs. One of these ORFs includes a signal peptide that is functional when fused to TnphoA.

Amino Acid Sequence↗
PubMed2000

Diversity of the HO gene encoding an endonuclease for mating-type conversion in the bottom fermenting yeast Saccharomyces pastorianus.

Two types of HO gene were cloned, sequenced and characterized from the bottom fermenting yeast Saccharomyces pastorianus. The HO gene present on the 1500 kb chromosome was designated Sc-HO (S. cerevisiae-type HO), because the nucleotide sequence of its promoter region and the open reading frame (ORF) was almost identical to that of the S. cerevisiae laboratory strain HO gene (Lab-HO). The other HO gene, designated Lg-HO (Lager-fermenting-yeast specific HO), showed 64% and 83% homology with the promoter and ORF of the Lab-HO at the nucleotide sequence level, respectively, and was located on the 1100 kb chromosome. Analysis of the 4 kb DNA fragment amplified from S. bayanus type strain indicated that the nucleotide sequence of S. bayanus-HO is almost identical to that of the Lg-HO. The SSB1 gene located downstream of the HO gene in S. cerevisiae was also found in the 3' distal region of the Sc-HO, Lg-HO and S. bayanus HO genes. These results showed that the genetic arrangement around the HO loci both of S. pastorianus and S. bayanus is identical to S. cerevisiae. Southern analysis has revealed that Saccharomyces sensu stricto contain four types of HO genes; S. paradoxus-type HO, the Sc-HO, the Lg-HO and S. uvarum-type HO genes. This HO gene diversity provides useful information for the classification of strains belonging to Saccharomyces sensu stricto. The S. pastorianus Sc-HO, Lg-HO and S. bayanus-HO Accession Nos in the DDBJ Nucleotide Sequence Database are AB027449, AB027450 and AB027451, respectively.

Amino Acid Sequence↗
PubMed1997

Genetic organization and transposition properties of IS511.

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobacter crescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISR1, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5' end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G + C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family.

Amino Acid Sequence↗
PubMed1999

DNA sequence of the beta-isopropylmalate dehydrogenase gene and phylogenetic analysis of the yeast Saccharomyces exiguus Yp74L-3.

The beta-isopropylmalate dehydrogenase (LEU2) gene from a homothallic wild-type yeast, Saccharomyces exiguus Yp74L-3, was analyzed to estimate the phylogenetic position of this strain in yeasts. The beta-isopropylmalate dehydrogenase gene of Yp74L-3 was first isolated as a clone complementing the leu2 mutation of Saccharomyces cerevisiae, and then confirmed to complement the haploid leu2 mutant derived from strain Yp74L-3 through genetic transformation. The nucleotide sequence of the cloned DNA revealed an open reading frame (ORF) encoding the beta-isopropylmalate dehydrogenase composed of 365 amino acids. The beta-isopropylmalate dehydrogenase coding sequence from the Yp74L-3 strain displayed 76.7% similarity to that of S. cerevisiae. Candidates for a UAS and a TATA-box in the 5'-upstream region and for a poly-A attachment site in the 3'-downstream region were found. A phylogenetic tree constructed from the nucleotide sequences of the beta-isopropylmalate dehydrogenase coding regions revealed that Yp74L-3 is located between S. cerevisiae and the Kluyveromyces yeasts. The LEU2 gene cloned from Yp74L-3 will serve as an effective genetic marker for constructing the transformation system in S. exiguus Yp74L-3.

Journal Article↗
PubMed1992

Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D.

From a genomic library of Chromatium vinosum strain D in lambda L47, a 16.5-kbp EcoRI-restriction fragment was identified by hybridization with a DNA fragment harboring the operon for Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthesis. This fragment and subfragments thereof restored the ability to synthesize and accumulate PHA in PHA-negative mutants of A. eutrophus. A region of 6977 bp was sequenced; seven open reading frames (ORFs) were identified which probably represent coding regions; six of these are most probably relevant for PHA biosynthesis in C. vinosum. The structural genes for biosynthetic acetyl-CoA acyltransferase (beta-ketothiolase; phbACv, 1188 bp) and NADH-dependent acetoacetyl-CoA reductase (phbBCv, 741 bp) were separated by ORF4 (462 bp) and ORF5 (369 bp). Downstream of phbBCv ORF7 (471 pb) was identified which was not completed at the 3' terminus. The functions of ORF4, ORF5, and ORF7 are not known. The amino acid sequences of beta-ketothiolase and acetoacetyl-CoA reductase deduced from phbACv and phbBCv, exhibited a similarity of 68.2% and 56.4% identical amino acids, respectively, to the corresponding enzymes of A. eutrophus. Antilinear to and upstream of the genes mentioned above, two genes were identified which were transcribed from a sigma 70-dependent promoter. This promoter overlapped with and was divergent to the phbACv promoter; the transcriptional start sites were mapped by S1 nuclease protection assays. These genes were ORF2 (1074 bp), whose function is not known but whose presence in Escherichia coli is essential for expression of PHA synthase activity, and the structural gene for a PHA synthase of low M(r) (phbCCv, 1068 bp). The gene products of ORF2 and phbCCv, with M(r) of 40,525 and 39,730, respectively, were expressed in E. coli applying the T7 RNA polymerase/promoter system. Although the amino acid sequence of PHA synthase deduced from phbCCv exhibited only 24.7% overall similarity with the PHA synthase of A. eutrophus, highly conserved regions were identified.

Acetyl-CoA C-Acyltransferase↗
PubMed2003

Molecular architecture of the regulatory Locus sae of Staphylococcus aureus and its impact on expression of virulence factors.

We characterized the sae operon, a global regulator for virulence gene expression in Staphylococcus aureus. A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity. Sequence analysis of the sae operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS. Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis. The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS. The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR. T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. sae-specific transcripts were detectable in all of the 40 different clinical S. aureus isolates investigated. Transcript levels were at maximum during the post-exponential growth phase. The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA. The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments. In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S. aureus.

Adhesins, Bacterial↗
PubMed2000

Characterization of a DNA region containing 5'-(CAAT)(n)-3' DNA sequences involved in lipooligosaccharide biosynthesis in Haemophilus somnus.

Repetitive tetranucleotide sequences of 5'-(CAAT)(n)-3' have been identified at the 5' end of an open reading frame (ORF) named lob1 from Haemophilus somnus strain 738. Based on sequence analysis, lob1 has 59% DNA homology to lex2B, which is involved in lipooligosaccharide (LOS) biosynthesis in H. influenzae. We now report that the number of 5'-CAAT-3' repeats in lob1 varied from 31-35, but that 94% of colonies contained 33 repeats of 5'-CAAT-3' downstream of two potential start codons, as determined by DNA sequence analysis of the 5'-CAAT-3' region from individual colonies. If transcription began with the start codon closest to the 5'-CAAT-3' repeats, a protein of 34.5 kDa would be encoded when 33 repeats were present. However, we could not establish a correlation between the number of 5'-CAAT-3' repeats in lob1 with a specific LOS electrophoretic profile or reactivity with two LOS monoclonal antibodies, indicating multiple genes control LOS phase variation in H. somnus. Complementation of strain 129Pt with lob1 containing 33 5 '-CAAT-3' repeats in shuttle vector pLS88 resulted in transformants 129Pt(pLSlob1-33A) and 129Pt(pLSlob1-33B), both of which demonstrated the same altered LOS electrophoretic profile. Unlike strain 129Pt, both transformants underwent limited LOS phase variation, which correlated with variation in the number of 5'-CAAT-3' repeats in pLSlob1-33. Nanoelectrospray-mass spectrometry of O-deacylated LOS indicated that transformant 129Pt(pLSlob1-33A) LOS was composed of a different distribution of glycoforms than LOS of the parent strain. The ratio of glucose to galactose changed from 1:2 in strain 129Pt LOS to 2:1 in transformant 129Pt(pLSlob1-33A) LOS, as determined by gas chromatography-mass spectrometry. Nuclear magnetic resonance spectroscopy confirmed and extended these observations. Transformant 129Pt(pLSlob1-33A) was constitutively more reactive in colony immunoblotting to polyclonal antiserum made to purified strain 738 LOS, and was more susceptible to complement-mediated killing in the presence of anti-738 LOS serum than parent strain 129Pt. Based on these results, Lob1 appears to be a phase variable galactosyl transferase involved in LOS biosynthesis in H. somnus.

Animals↗
PubMed2003

Regulation of fungal gene expression via short open reading frames in the mRNA 5'untranslated region.

We review how the expression of fungal mRNAs can be controlled by ribosome interactions with short upstream open reading frames (uORFs) within the 5'untranslated region. The efficiency of uAUG recognition modulates the impact of a uORF but steps during and after translation of the uORF also influence uORF function. The post-termination behaviour of ribosomes, therefore, plays a major role in determining the expression level of these main ORFs. Translation of a uORF can produce a cis-acting peptide that causes effector molecule-dependent stalling of the ribosomes at the end of the uORF. In other cases it is the length or position, or other features of the uORF, rather than the peptide it encodes, that determine the efficiency with which ribosomes reinitiate translation downstream of it. Whether the form of the ribosome that resumes scanning after termination is the 40S subunit alone or the entire 80S ribosome is not known. Translation of the uORF can also control gene expression by affecting the stability of the mRNA. Finally, trans-acting factors may participate in the regulatory mechanisms. Future work will need not only to provide more information on the mechanisms underlying the known cases of uORF-mediated control but also to define the full complement of uORF-containing mRNAs in at least one fungal organism.

5' Untranslated Regions↗
PubMed2006

Antisense-induced ribosomal frameshifting.

Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1-ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem-loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5' of the shift site and to polyamine levels.

Animals↗
PubMed2000

Purification and molecular characterization of a tripeptidase (PepT) from Lactobacillus helveticus.

A tripeptidase (PepT) from a thermophilic dairy starter strain of Lactobacillus helveticus was purified by four chromatographic steps. PepT appeared to be a trimeric metallopeptidase with a molecular mass of 150 kDa. PepT exhibited maximum activity against hydrophobic tripeptides, with the highest activity for Met-Gly-Gly (K(m), 2.6 mM; V(max), 80.2 micromol. min(-1). microg(-1)). Some of the hydrophobic dipeptides were slowly hydrolyzed, distinguishing the Lactobacillus PepT from its counterpart in mesophilic Lactococcus lactis. No activity against tetrapeptides or amino acid p-nitroanilide derivatives was observed. The pepT gene and its flanking regions were isolated by PCR and sequenced by cyclic sequencing. The sequence analyses revealed open reading frames (ORFs) 816 bp (ORF1) and 1,239 bp (ORF2) long. ORF2 encoded a 47-kDa PepT protein which exhibited 53% identity with the PepT from L. lactis. The mRNA analyses indicated that pepT conforms a novel operon structure with an ORF1 located upstream. Several putative -35/-10 regions preceded the operon, but only one transcription start site located downstream of the first putative -10 region was identified. An inverted repeat structure with DeltaG of -64.8 kJ/mol was found downstream of the PepT-encoding region.

Amino Acid Sequence↗
PubMed1992

Transcription from varicella-zoster virus gene 67 (glycoprotein IV).

Three transcripts map to the varicella-zoster virus (VZV) open reading frame (ORF) 67, which encodes glycoprotein IV (gpIV). All of these transcripts are polyadenylated and are transcribed from left to right towards the genomic terminal short repeats. Previous Northern (RNA) blot analyses suggested that the most abundant of these transcripts (1.65 kb) might code for gpIV. We performed S1 nuclease protection and primer extension assays and determined that the 5' terminus of the 1.65-kb transcript maps 91 bp upstream from the gpIV initiation codon. An AT-rich region (ATAAA), -28 bp from the cap site, is a potential TATA box, and at -71 bp there is a consensus CCAAT box motif. The 3' end of the 1.65-kb transcript is 20 bp downstream of two overlapping polyadenylation signals, AATAAA and ATTAAA, and just downstream of the 3' terminus is a GU-rich sequence. These results are reminiscent of data from our analysis of the VZV gpV gene, confirming that VZV appears able to use unusual TATA box motifs. Many canonical TATA sequences are present upstream from these VZV transcriptional start sites but, apparently, are not used. We tested sequences upstream from the gpIV cap site for promoter activity in transient expression experiments by cloning a DNA fragment (+63 to -343 bp) into pCAT3M, which contains a chloramphenicol acetyltransferase reporter gene. This clone showed little constitutive promoter activity but was activated more than 200-fold by infection with VZV and 5-fold with herpes simplex virus. The two known VZV transactivating genes (those for ORF 4 and ORF 62) were tested for their abilities to activate expression from the gpIV promoter by using their cognate promoters. The ORF 4 gene was minimally active, whereas the ORF 62 gene gave twofold induction; both genes, acting together, gave fivefold induction. However, replacement of the IE62 promoter with the immediate-early cytomegalovirus promoter in the ORF 62 construct gave over 40-fold induction of chloramphenicol acetyltransferase activity under the gpIV promoter in the same assay.

Amino Acid Sequence↗
PubMed2000

New breakpoints in both the H4 and RET genes create a variant of PTC-1 in a post-Chernobyl papillary thyroid carcinoma.

Two main types of RET/PTC oncogene, named RET/PTC-1 and 3, occur in papillary thyroid carcinomas especially in those from Belarus children after the Chernobyl nuclear accident. Several variants of RET/PTC-3 have also been found, having different break points with respect to the classical RET/PTC-3. To our knowledge, no variant of RET/PTC-1 has been described up to now. We found a post-Chernobyl papillary thyroid carcinoma with an RET/PTC-1 rearrangement characterized by a transcript longer than expected. Sequence analysis of the PCR product obtained after RT-PCR revealed new fusion points between H4 and RET genes. The genomic sequence showed new breakpoints in both H4 intronic and in RET exonic regions. The RET gene breakpoint occurred within exon 11, at variance with the classical form of RET/PTC-1, in which it is in intron 11. As a consequence of this new fusion point, the transcript included 132 nucleotides of exon 11, coding for 44 amino acids of RET protein. Regarding the H4 gene, the classical breakpoint is in the first intron and the cDNA contains a fragment of 339 nucleotides. In our case the cDNA had a longer fragment of H4 involving a total of 1266 nucleotides. Sequencing of genomic DNA revealed a rearrangement breakpoint at position 886 of a new H4 intron located downstream of the 1266 coding region. Furthermore, as a consequence of the activation of a cryptic splicing site, 132 nucleotides of this intron were spliced between the H4 and RET genes. Sequence analysis of the new chimera showed that the original frames of H4 and RET were joint with the intronic sequence without disruption of the open reading frame (ORF). Moreover, the genomic DNA of this case showed transforming activity in the DNA-mediated transfection assay using NIH-3T3 cells. In conclusion, we describe here the first variant of RET/PTC-1 oncogene, which we have termed 'long'-PTC-1, characterized by new breakpoints of both genes involved in the rearrangement and having transforming activity. Similar to previously reported PTC-3 variants, long-PTC-1 has been found in a post-Chernobyl papillary thyroid carcinoma confirming that RET/PTC rearrangements other than the classical forms (RET/PTC-1 and -3) are specifically associated with radiation-induced papillary thyroid cancer.

Carcinoma, Papillary↗
PubMed1999

The spectrum of mutations in TBX3: Genotype/Phenotype relationship in ulnar-mammary syndrome.

Ulnar-mammary syndrome (UMS) is a pleiotropic disorder affecting limb, apocrine-gland, tooth, hair, and genital development. Mutations that disrupt the DNA-binding domain of the T-box gene, TBX3, have been demonstrated to cause UMS. However, the 3' terminus of the open reading frame (ORF) of TBX3 was not identified, and mutations were detected in only two families with UMS. Furthermore, no substantial homology outside the T-box was found among TBX3 and its orthologues. The subsequent cloning of new TBX3 cDNAs allowed us to complete the characterization of TBX3 and to identify alternatively transcribed TBX3 transcripts, including one that interrupts the T-box. The complete ORF of TBX3 is predicted to encode a 723-residue protein, of which 255 amino acids are encoded by newly identified exons. Comparison of other T-box genes to TBX3 indicates regions of substantial homology outside the DNA-binding domain. Novel mutations have been found in all of eight newly reported families with UMS, including five mutations downstream of the region encoding the T-box. This suggests that a domain(s) outside the T-box is highly conserved and important for the function of TBX3. We found no obvious phenotypic differences between those who have missense mutations and those who have deletions or frameshifts.

Abnormalities, Multiple↗
PubMed2001

Biosynthetic pathway of cephabacins in Lysobacter lactamgenus: molecular and biochemical characterization of the upstream region of the gene clusters for engineering of novel antibiotics.

The cephabacins, one of the beta-lactam antibiotics, are produced by Lysobacter lactamgenus. The previous studies the cephabacin biosynthesis were limited to a gene cluster that encodes the gene products responsible for the biosynthesis of the cephem nucleus. The long-term goal of this research is to elucidate the metabolic diversity and biosynthetic pathway of cephabacins and to design and/or discover new pharmacologically active compounds by engineering the cephabacin biosynthetic pathway in L. lactamgenus. In this study, we have cloned and sequenced a 24-kb fragment of a DNA locus upstream of the previously reported but incomplete putative ORF9 of L. lactamgenus. This contains three putative ORFs (the complete ORF9, ORF10, and ORF11) transcribed in the same direction and one putative ORF (ORF12) in the opposite direction. The isolated DNA locus extends the previously cloned part of the DNA locus containing the genes responsible for biosynthesis of the cephem nucleus up to 45 kb. The 42-kb fragment of the 45-kb gene cluster is located between a potential TATA box just upstream of the ORF11 and a termination loop just downstream of the previously reported bla gene. The complete ORF9 contains three nonribosomal peptide synthetase (NRPS) modules and one polyketide synthase (PKS) module and the ORF11 contains one NRPS module. The complete ORF9 also contains a putative thioesterase domain at the C-terminal end. We predicted the amino acid specificity of the four NRPSs by generating specificity binding pockets and expressed one of the NRPSs to confirm the amino acid specificity. The adenylation domain of the NRPS1, which is the last module of the NRPSs, showed significant amino acid specificity for L-arginine. These findings are in perfect agreement with the composition that was expected for the structure of cephabacins which contain an acetate residue, an L-arginine, and one to three L-alanines at the C-3' position of the cephem nucleus of cephabacins. The ORF10, encoding a putative ABC transporter which might be involved in conferring resistance against cephabacins, was identified between the complete ORF9 and the ORF11. Therefore, the complete ORF9, ORF10, ORF11 reported here and the other genes previously reported constitute an operon for the biosynthesis of cephabacins in L. lactamgenus. Based on our results, the biosynthetic pathways of acetate and elongated peptide moieties and a mechanism by which cephabacins are assembled by connecting the peptide moiety synthesized by the gene products of the complete ORF9 and the ORF11 to the C-3' position of the cephem nucleus synthesized by the gene products of pcbAB, pcbC, cefE, cefF, and cefD have been elucidated.

Amino Acid Sequence↗
PubMed2000

Genetic organisation of the M protein region in human isolates of group C and G streptococci: two types of multigene regulator-like (mgrC) regions.

In addition to beta-haemolytic streptococci belonging to Lancefield group A (Streptococcus pyogenes, GAS), human isolates of group C (GCS) and group G (GGS) streptococci (S. dysgalactiae subsp. equisimilis) have been implicated as causative agents in outbreaks of purulent pharyngitis, of wound infections and recently also of streptococcal toxic shock-like syndrome. Very little is known about the organisation of the genomic region in which the emm gene of GCS and GGS is located. We have investigated the genome sequences flanking the emm gene in GCS by sequencing neighbouring fragments obtained by inverse PCR. Our sequence data for GCS strains 25287 and H46A revealed two types of arrangement in the emm region, which differ significantly from the known types of mga regulon in GAS. We named this segment of the genome mgrC (for multigene regulon-like segment in group C streptococci). In strains belonging to the first mgrC type (prototype strain 25287) the emm gene is flanked up-stream by mgc, a gene that is 61% identical to the mga gene of GAS. A phylogenetic analysis of the deduced protein sequences showed that Mgc is related to Mga proteins of various types of GAS but forms a distinct cluster. Downstream of emm, the mgrC sequence region is bordered by rel. This gene encodes a protein that functions in the synthesis and degradation of guanosine 3',5' bipyrophosphate (ppGpp) during the stringent regulatory response to amino acid deprivation. In the second mgrC type (prototype strain H46A), the genes mgc and emm are arranged as in type 1. But an additional ORF (orf) is inserted in opposite orientation between emm and rel. This orf shows sequence homology to cpdB, which is present in various microorganisms and encodes 2',3' cyclo-nucleotide 2'-phosphodiesterase. PCR analysis showed that these two mgrC arrangements also exist in GGS. Our sequence and PCR data further showed that both types of mgrC region in GCS and GGS are linked via rel to the streptokinase region characterised recently in strain H46A. A gene encoding C5a peptidase, which is present at the 3' end of the mga regulon in GAS, was not found in the mgrC region identified in the GCS and GGS strains investigated here.

Amino Acid Sequence↗
PubMed2004

Ssn6-Tup1 requires the ISW2 complex to position nucleosomes in Saccharomyces cerevisiae.

The Imitation SWItch (ISWI) chromatin remodeling factors have been implicated in nucleosome positioning. In vitro, they can mobilize nucleosomes bi-directionally, making it difficult to envision how they can establish precise translational positioning of nucleosomes in vivo. It has been proposed that they require other cellular factors to do so, but none has been identified thus far. Here, we demonstrate that both ISW2 and TUP1 are required to position nucleosomes across the entire coding sequence of the DNA damage-inducible gene RNR3. The chromatin structure downstream of the URS is indistinguishable in Deltaisw2 and Deltatup1 mutants, and the crosslinking of Tup1 and Isw2 to RNR3 is independent of each other, indicating that both complexes are required to maintain repressive chromatin structure. Furthermore, Tup1 repressed RNR3 and blocked preinitiation complex formation in the Deltaisw2 mutant, even though nucleosome positioning was completely disrupted over the promoter and ORF. Our study has revealed a novel collaboration between two nucleosome-positioning activities in vivo, and suggests that disruption of nucleosome positioning is insufficient to cause a high level of transcription.

Adenosine Triphosphatases↗