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Complete mitochondrial DNA sequence of a Conoidean gastropod, Lophiotoma (Xenuroturris) cerithiformis: gene order and gastropod phylogeny.

We have determined the first complete nucleotide sequence of the mitochondrial genome of a venomous mollusc, the Conoidean gastropod, Lophiotoma (Xenuroturris) cerithiformis. It is 15,380 nucleotide pairs (ntp) and encodes 13 proteins, two ribosomal RNAs and 22 tRNAs of the mitochondrion's own protein synthesizing system. The protein mRNAs, ribosomal RNAs and 13 of the tRNAs are transcribed from the same strand, the remaining tRNAs from the other strand. The longest segment of unassigned sequence is 139 ntp and includes a 82 ntp segment that is a perfect inverted repeat sequence of 37 ntp separated by 8 nt. The gene arrangement of L. cerithiformis mtDNA shows remarkable similarity to the gene arrangements of mtDNAs of the vetigastropod Haliotis rubra, the polyplacophoran Katharina tunicata and the cephalopod Octopus vulgaris, but differs dramatically from the gene arrangements found in the mtDNAs of pulmonate and opisthobranch gastropods, as well as mtDNAs of bivalves and scaphopods. A single sixteen gene inversion that distinguishes L. cerithiformis mtDNA from mtDNAs of H. rubra, K. tunicata and O. vulgaris is shared by mtDNA of a littorinomorph gastropod Littorina saxitalis, suggesting a close relationship of conoidean and littorinomorph gastropods.

Amino Acid Sequence↗

[Cloning, structure and features of a Saccharomyces cerevisiae DNA fragment causing the expression of reporter genes].

A DNA fragment "PX" of 240 b.p. was isolated at random from the genomic sequences of S. cerevisiae by using a plasmid that contained a promoterless reporter gene lacZ of E. coli. "PX" was capable of activating synthesis of three reporter genes (pho5 from yeast and bacterial lacZ and neo) in yeast cells bidirectionally. Results of Southern-blot hybridization with yeast genomic DNA suggest that the cloned sequence is represented in at least ten copies per cell. Nucleotide sequence of "PX" shows that DNA contains putative TATA elements and four tandems of inverted repeats: [sequence: see text].

Autoradiography↗

Cloning, molecular characterization and expression analysis of two small heat shock genes isolated from wine Lactobacillus plantarum.

AIM: Understanding the molecular response to stress tolerance of wine Lactobacillus plantarum. METHODS AND RESULTS: Two genes codifying for heat shock proteins were cloned from wine L. plantarum. The coding regions of the two heat shock genes are 420 and 444 nucleotides long, and started with an ATG codon suggesting that they were translated. The protein sequences deduced from the isolated genes have a molecular mass of 18.483 and 19.282 kDa, respectively, and were therefore named hsp18.5 and hsp19.3. The expression of small heat shock genes was analysed by RT-PCR analysis. Moreover, the 5' and 3' noncoding regions were cloned and sequenced. CONCLUSIONS: The expression of the heat shock genes was strongly induced by heat, cold and ethanol stress. Analysis of the 5' and 3' flanking regions of hsp18.5 and hsp19.3 genes, revealed the presence of an inverted repeat sequence (TTAGCACTC-N(9)-GAGTGCTAA) homologue to the CIRCE elements found to the upstream regulatory region of heat shock operons, and an inverted sequence that could form a stem and loop structure that it is likely to function as a transcriptional terminator. Based on their structures, the genes were classified as belonging to Class I of heat shock genes according to the B. subtilis nomenclature of heat response genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Small heat shock genes isolated from wine L. plantarum might have a role in preventing damage by cold stress.

Amino Acid Sequence↗

Characterization of foldback sequences in Physarum polycephalum nuclear DNA using the electron microscope.

An examination of the foldback fraction of nuclear DNA from Physarum polycephalum has been carried out using the electron microscope. Results show that the inverted repeat sequences responsible for the formation of foldback DNA range from 150-3000 bases in length, with a number-average size of 340 bases. About one-half of the inverted sequences form looped structures with loop sizes averaging 1200 bases in length. The distance between adjacent foldback sequences is estimated to be in the range 100-1500 bases.

Cell Nucleus↗

pEG plasmid involved in styrene degradation: molecular dimorphism and integration of a segment into the chromosome.

In the Pseudomonas fluorescens strain ST the ability to utilize styrene as the sole carbon source is due to the presence of a plasmid, pEG. In the present report we show that pEG contains two inverted repeat sequences and we present evidence indicating that the catabolic genes are localized in these repeats. The region separating the inverted repeats can assume alternative orientations. In the chromosome of strain ST, a 3 kbp region is homologous to sequences present at one end of the plasmid repeats. This region is present in one copy in the chromosome and could be a specific site for integration of the plasmid. We suggest that this sequence, which is present twice in the pEG plasmid and once in the chromosome, might be a transposon-like element.

Biodegradation, Environmental↗

Phenobarbital and sulfonylurea-inducible operons encoding herbicide metabolizing cytochromes P-450 in Streptomyces griseolus.

We have identified the promoters for two inducible genes, in Streptomyces griseolus, that encode herbicide-metabolizing cytochromes P-450. They are in the class of promoters that have -35 and -10 sequences similar to those used in Escherichia coli by RNA polymerase E sigma 70. Transcription from either promoter was shown to be induced by sulfonylurea (chlorimuron ethyl) or phenobarbital. Mapping of mRNA showed that each cytochrome P450-encoding gene was transcribed on a separate multicistronic mRNA that encodes cytochrome P-450 (suaC or subC), ferredoxin (suaB or subB) and at least one other open reading frame. An inducible, site-specific DNA-binding activity was identified that bound to two similar 8-bp inverted repeat sequences within or near the sua promoter (suaP). A noninducible DNA-binding activity, distinct from that which bound to suaP, was found that bound to an 11-bp inverted repeat at the sub transcription start point.

Base Sequence↗

The nucleotide sequence of maize streak virus DNA.

The nucleotide sequence of the DNA of maize streak virus (MSV) has been determined. The data were accommodated into one DNA circle of 2687 nucleotides, in contrast to previously characterised geminiviruses which have been shown to possess two circles of DNA. Comparison of the nucleotide sequences of the DNA of MSV with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) showed no detectable homology. Analysis of open reading frames revealed seven potential coding regions for proteins of mol. wt. greater than or equal to 10 000, three in the viral (+) sense and four in the complementary (-) sense. The position of likely transcription signals on the MSV DNA sequence would suggest a bidirectional strategy of transcription as proposed for CLV and TGMV. Nine inverted repeat sequences which have a potential of forming hairpin structures of delta G greater than or equal to -14 kcal/mol have been detected. Three of these hairpin structures are in non-coding regions and could be involved in the regulation of transcription and/or replication.

Base Sequence↗

Organization of ribosomal protein genes rpl23, rpl2, rps19, rpl22 and rps3 on the Euglena gracilis chloroplast genome.

The nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rpl23-150 bp spacer-rpl2-59 bp spacer-rps19-110 bp spacer-rpl22-630 bp spacer-rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rpl23, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rpl2 and rpl22 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53-76% T, 25-34% A, 3-10% G and 2-7% C in the mRNA-like strand. These six introns show minimal resemblance to group II chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5' and 3' ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rps19 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18-50%, and 29-58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rpl23 and downstream from rps3.

Amino Acid Sequence↗

A novel integron-like element carrying the metallo-beta-lactamase gene blaIMP.

A plasmid-mediated metallo-beta-lactamase gene was cloned from a carbapenem-resistant Serratia marcescens strain, AK9373. The metallo-beta-lactamase gene was identical to the blaIMP, and it was located in the space between an integrase-like gene and an aac(6')-Ib-like gene. The deduced amino acid sequence for the putative integrase gene showed considerable identity (60.9%) to that of the Escherichia coli integrase reported. Sequences similar to the GTTRRRY and an atypical 59-base element containing a 67-bp inverted repeat sequence, which were peculiar to the integrase-dependent recombination, were also conserved in the flanking regions of the blaIMP gene. These findings imply that the metallo-beta-lactamase gene in S. marcescens AK9373 is carried by a novel integron-like element that is mediated by a transferable large plasmid.

Amino Acid Sequence↗

Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids.

By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm). Plasmid NTP1, which mediates Ap resistance, contains Tn1701. Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm). The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.

Ampicillin↗

Identification and distribution of new insertion sequences in the genome of alkaliphilic Bacillus halodurans C-125.

Fifteen kinds of new insertion sequences (ISs), IS641 to IS643, IS650 to IS658, IS660, IS662, and IS663, and a group II intron (Bh.Int) were identified in the 4,202,352-bp genome of alkaliphilic Bacillus halodurans C-125. Out of 120 ISs identified in the C-125 genome, 29 were truncated, indicating the occurrence of internal rearrangements of the genome. The ISs other than IS650, IS653, IS660, and IS663 generated a 2- to 9-bp duplication of the target site sequence, and the ISs other than IS650, IS653, and IS657 carry 14- to 64-bp inverted repeats. Sequence analysis revealed that six kinds of ISs (IS642, IS643, IS654, IS655, IS657, and IS658) belong to a separate IS family (IS630, IS21, IS256, IS3, IS200/IS605, and IS30, respectively) as a new member. Also, IS651 and IS652 were characterized as new members of the ISL3 family. Significant similarity was found between the transposase (Tpase) sequences between IS650 and IS653 (78.2%), IS651 and IS652 (56.3%), IS656 and IS662 (71.0%), and IS660 and IS663 (44.5%), but the others showed no similarity to one another. Tpases in 28 members of IS651 in the C-125 genome were found to have become diversified. Most of the IS elements widely distributed throughout the genome were inserted in noncoding regions, although some genes, such as those coding for an ATP-binding cassette transporter/permease, a response regulator, and L-indole 2-dehydrogenase, have been mutated through the insertion of IS elements. It is evident, however, that not all IS elements have transposed and caused rearrangements of the genome in the past 17 years during which strain C-125 was subcultured under neutral and alkaline conditions.

Bacillus↗

Periodic organisation of foldback sequences in Physarum polycephalum nuclear DNA.

Nuclear DNA from the slime mould Physarum polycephalum is shown to contain interspersed inverted repeat sequences, such that denatured fragments of DNA containing pairs of these sequences form intra-chain duplexes under appropriate conditions. The organisation and distribution of the nucleotide sequences responsible for the formation of foldback structures in Physarum DNA have been investigated using the electron microscope. The majority of foldback duplexes have sizes ranging up to 800 base pairs, and about 60-80% of DNA molecules 2.2 X 10(4) bases in length contain interspersed foldback elements. The size of individual foldback duplexes, and also the length of the intervening sequences which separate them, are non-random. The results can best be explained by a model in which separate foldback foci in Physarum DNA are spaced periodically at regular intervals. The regions containing foldback foci are thought to contain smaller, tandemly-arranged sequences of discrete sizes, in some cases related to other nucleotide sequences of a similar nature in the same locality in Physarum DNA.

Base Sequence↗

Archaebacteria: transcription and processing of ribosomal RNA sequences in Halobacterium cutirubrum.

The chromosome of Halobacterium cutirubrum contains a single ribosomal RNA gene cluster. The 5' to 3' organization of genes within this 6-kpb region is: 16S, alanine tRNA, 23S, 5S, cysteine tRNA. The entire gene cluster is transcribed as a single long primary transcript; processing of mature RNA sequences from the 5' region of the transcript begins prior to the completion of synthesis at the 3' end. There are five conserved octanucleotide direct repeats (TGCGAACG) in the 900-bp 5'-flanking sequence in front of the 16S gene. The positions of these repeat sequences correspond to the different 5' ends of the primary transcript and probably represent the RNA polymerase start sites. The 16S and 23S rRNA genes are surrounded by long nearly perfect inverted repeat sequences. These sequences probably form duplex structures in the primary transcript and are recognized by an RNaseIII-like endonuclease activity that carries out the initial excision of the precursor 16S and 23S rRNA sequences. These precursors are rapidly trimmed tot he mature 16S and 23S molecules and assembled into ribosomal particles. The processing sites for 5S rRNA appear to be at or very near to the mature ends of the 5S molecule. The tRNA sequences are processed with reduced efficiency from the primary transcript. Nuclease cuts have been detected at the ends as well as in the middle of the cysteine tRNA sequence suggesting that there may be alternative processing pathways, one resulting in proper excision of the mature tRNA sequence and the other resulting in improper excision and degradation of the tRNA sequence. The transcription termination sequence is believed to be at or beyond an AT-rich sequence preceded by a GC-rich sequence located distal to the cysteine tRNA gene.

Base Sequence↗

Transposition of Tnr1 in rice genomes to 5'-PuTAPy-3' sites, duplicating the TA sequence.

Tnr1 is a repetitive sequence in rice with several features characteristic of a transposable DNA element. Its copy number was estimated to be about 3500 per haploid genome by slot-blot hybridization. We have isolated six members of Tnr1 located at different loci by PCR (polymerase chain reaction) and determined their nucleotide sequences. The Tnr1 elements were similar in size and highly homologous (about 85%) to the Tnr1 sequence identified first in the Waxy gene in Oryza glaberrima. A consensus sequence of 235 bp could be derived from the nucleotide sequences of all the Tnr1 members. The consensus sequence showed that base substitutions occurred frequently in Tnr1 by transition, and that Tnr1 has terminal inverted repeat sequences of 75 bp. Almost all the chromosomal sequences that flank the Tnr1 members were 5'-PuTA-3' and 5'-TAPy-3', indicating that Tnr1 transposed to 5'-PuTAPy-3' sites, duplicating the TA sequence. PCR-amplified fragments from some rice species did not contain the Tnr1 members at corresponding loci. Comparison of nucleotide sequences of the fragments with or without a Tnr1 member confirmed preferential transposition of Tnr1 to 5'-PuTAPy-3' sites, duplicating the TA sequence. One amplified sequence suggested that imprecise excision had occurred to remove a DNA segment containing a Tnr1 member and its neighboring sequences at the Waxy locus of rice species with genome types other than AA. We also present data that may suggest that Tnr1 is a defective form of an autonomous transposable element.

Adenine Nucleotides↗

Cloning and sequencing of two chromosomal lipase genes from Geotrichum candidum.

Two chromosomal lipase genes (lip1 and lip2 encoding lipases I and II, respectively) were cloned from Geotrichum candidum by colony hybridization using lipase cDNAs as probes, and their nucleotide sequences were determined. Both genes contained 5'- and 3'-flanking regions in addition to their coding regions. The coding regions contained no intron, and there was 86% homology between their nucleotide sequences. However, no homology was observed between the flanking regions. The primer extension analysis showed that four transcription initiation sites were localized around 100-bp upstream from the ATG start codon of the lip2 gene. The putative promoter sequences of eukaryotic genes, CCAAT-----TATAAA, were found in the 5'-flanking regions of two lipase genes. These sequences might participate in the lipase induction by long-chain fatty acid. There were two inverted repeat sequences and a pyrimidine-rich sequence in the 5'-flanking region of lip2, but not in the 5'-flanking region of lip1. This specific structure of lip2 might reflect the higher transcriptional efficiency.

Amino Acid Sequence↗

An unusual Tn21-like transposon containing an ars operon is present in highly arsenic-resistant strains of the biomining bacterium Acidithiobacillus caldus.

A transposon, TnAtcArs, that carries a set of arsenic-resistance genes was isolated from a strain of the moderately thermophilic, sulfur-oxidizing, biomining bacterium Acidithiobacillus caldus. This strain originated from a commercial plant used for the bio-oxidation of gold-bearing arsenopyrite concentrates. Continuous selection for arsenic resistance over many years had made the bacterium resistant to high concentrations of arsenic. Sequence analysis indicated that TnAtcArs is 12 444 bp in length and has 40 bp terminal inverted repeat sequences and divergently transcribed resolvase and transposase genes that are related to the Tn21-transposon subfamily. A series of genes consisting of arsR, two tandem copies of arsA and arsD, two ORFs (7 and 8) and arsB is situated between the resolvase and transposase genes. Although some commercial strains of At. caldus contained the arsDA duplication, when transformed into Escherichia coli, the arsDA duplication was unstable and was frequently lost during cultivation or if a plasmid containing TnAtcArs was conjugated into a recipient strain. TnAtcArs conferred resistance to arsenite and arsenate upon E. coli cells. Deletion of one copy of arsDA had no noticeable effect on resistance to arsenite or arsenate in E. coli. ORFs 7 and 8 had clear sequence similarity to an NADH oxidase and a CBS-domain-containing protein, respectively, but their deletion did not affect resistance to arsenite or arsenate in E. coli. TnAtcArs was actively transposed in E. coli, but no increase in transposition frequency in the presence of arsenic was detected. Northern hybridization and reporter gene studies indicated that although ArsR regulated the 10 kb operon containing the arsenic-resistance genes in response to arsenic, ArsR had no effect on the regulation of genes associated with transposition activity.

Acidithiobacillus↗

Repression and catabolite repression of the lactose operon of Staphylococcus aureus.

The lacR gene encodes the repressor of the lactose operon of S. aureus. The nucleotide sequence of this gene and the promoter-operator region of the operon are reported. The lacR gene encodes a protein with a molecular weight of 28,534. This protein was found to share sequence homology with the DeoR protein, the repressor of the E. coli deoxyribonucleotide operon. Directly and invertedly repeated sequences were found associated with the promoter for the structural genes of the operon. These sequences were examined by site-directed mutagenesis and found to be important in repressor binding and in the binding of a catabolite repressor. Evidence is presented in support of a model for catabolite repression of the operon which involves a negative-acting transcriptional regulator which binds to the promoter region of the operon and prevents transcription.

Amino Acid Sequence↗

The very large amplifiable element AUD2 from Streptomyces lividans 66 has insertion sequence-like repeats at its ends.

In a spontaneous, chloramphenicol-sensitive (Cms), arginine-auxotrophic (Arg-) mutant of Streptomyces lividans 1326, two amplified DNA sequences were found. One of them was the well-characterized 5.7-kb ADS1 sequence, amplified to about 300 copies per chromosome. The second one was a 92-kb sequence called ADS2. ADS2 encoding the previously isolated mercury resistance genes of S. lividans was amplified to around 20 copies per chromosome. The complete ADS2 sequence was isolated from a genomic library of the mutant S. lividans 1326.32, constructed in the phage vector lambda EMBL4. In addition, the DNA sequences flanking the corresponding amplifiable element called AUD2 in the wild-type strain were isolated by using another genomic library prepared from S. lividans 1326 DNA. Analysis of the ends of AUD2 revealed the presence of an 846-bp sequence on both sides repeated in the same orientation. Each of the direct repeats ended with 18-bp inverted repeated sequences. This insertion sequence-like structure was confirmed by the DNA sequence determined from the amplified copy of the direct repeats which demonstrated a high degree of similarity of 65% identity in nucleic acid sequence to IS112 from Streptomyces albus. The recombination event leading to the amplification of AUD2 occurred within these direct repeats, as shown by DNA sequence analysis. The amplification of AUD2 was correlated with a deletion on one side of the flanking chromosomal region beginning very near or in the amplified DNA. Strains of S. lividans like TK20 and TK21 which are mercury sensitive have completely lost AUD2 together with flanking chromosomal DNA on one or both sides.

Amino Acid Sequence↗