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A map of the restriction targets in yeast 2 micron plasmid DNA cloned on bacteriophage lambda.

The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage lambda. The two forms of circular DNA which exist in equilibrium due to recombination between inverted repeat sequences were separated as stable clones, and a map of targets for restriction endonucleases EcoRI, HindIII and HpaI was constructed. The circular DNAs isolated from a particular oligomycin resistant strain and its parent oligomycin snesitive strain were compared by restriction endonuclease analysis, and no difference was detected. The potential uses of cloned 2 micron DNA in determining the possible biological role of these plasmids are considered.

Coliphages↗

Mitochondrial genome of the dimorphic zygomycete Mucor racemosus.

Mitochondria were isolated from the dimorphic zygomycete Mucor racemosus by differential centrifugation. DNA from the organelles was purified by cesium chloride-ethidium bromide isopycnic centrifugation. Examination of the mitochondrial DNA by electron microscopy revealed a circular chromosome approximately 63.8 kbp in circumference. The chromosome was digested with restriction endonucleases and the resulting DNA fragments were separated by agarose-gel electrophoresis. Electophoretic mobilities and stoichiometry of the fragments indicated a mixed population of mtDNA molecules each with a size of about 63.4 kbp. Physical maps were constructed from analyses of fragments generated in single and double restriction digests and from the hybridization of fragments to probes for the large and small mitochondrial rRNA genes from Saccharomyces cerevisiae. The Mucor mitochondrial chromosome was found to exist in the form of two flip-flop isomers with inverted repeat sequences encoding both rRNA genes.

Cloning, Molecular↗

Tn2610, a transposon involved in the spread of the carbenicillin-hydrolyzing beta-lactamase gene.

We have found a new transposon, Tn2610, on pCS200 in clinical isolates of Escherichia coli, which encodes the carbenicillin-hydrolyzing beta-lactamase gene in combination with the resistance determinants to streptomycin and sulfonamide. Tn2610 has a molecular size of 24 kilobase pairs and is flanked by long inverted repeat sequences of 3 kilobase pairs in length. Genetical and physical analyses indicate that Tn2610 is a single transposable unit encoding the multiple resistance determinants and that is different from any previously described transposon. The characteristic DNA structure observed in various complex resistance transposons involved in the transposition of the carbenicillin-hydrolyzing beta-lactamase gene is discussed.

Carbenicillin↗

Regulation of Mu element copy number in maize lines with an active or inactive Mutator transposable element system.

In the progeny of an active Mutator plant, the number of Mu elements increases on self-pollination and maintains the average parental Mu content on outcrossing to a non-Mutator line; both patterns of transmission require an increase in the absolute number of Mu elements from one generation to the next. The same average copy number of Mu elements is transmitted through the male and female, but there is wide variation in the absolute copy number among the progeny. In inactive Mutator plants-defined both by the loss of somatic instability at a reporter gene (bronze2-mu1) and by modification of the HinfI sites in the terminal inverted repeat sequences of Mu elements - the absolute copy number of Mu elements is fixed in the parent. Thus, in outcrosses Mu element number is halved, and on self-pollination Mu copy number is constant. Reactivation of somatic mutability at cryptic bz2-mu1 alleles in inactive individuals by crossing to an active line seems not to involve an increase in Mu element copy number transmitted by the inactive individual. These and other results suggest that increases in Mu copy number occur late in plant development or in the gametophyte rather than after fertilization.

DNA Transposable Elements↗

Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI restriction fragments of bacteriophage P1 DNA.

A cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases. The enzymes used and, in parenthesis, the number of their cleavage sites on the P1clts genome are: PstI (1), HindIII(3), BglII (11), BamHI (14) and EcoRI (26). The relative order of the PstI, HindIII and BglII sites, as well as the order of 13 out of the 14 BamHI sites and of 17 out of the 26 EcoRI sites was determined. The P1 genome was divided into 100 map units and the PstI site was arbitrarily chosen as reference point at map unit 20. DNA packaging into phage heads starts preferentially at map unit 92 and it proceeds towards higher map units. The two inverted repeat sequences of P1 DNA map about at units 30 and 34.

Base Sequence↗

The 3' untranslated regions of chloroplast genes in Chlamydomonas reinhardtii do not serve as efficient transcriptional terminators.

A general characteristic of the 3' untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure. These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3' end processing signals in spinach chloroplasts, and in the atpB mRNA of Chlamydomonas reinhardtii chloroplasts. In order to carry out a broad study of the efficiency of the untranslated sequences at the 3' ends of chloroplast genes in Chlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments using Chlamydomonas chloroplast transformants in which different 3' ends were inserted into the chloroplast genome between a petD promoter and a reporter gene. The results showed that none of the 3' ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators. Therefore, we suggest that most or all of the 3' ends of mature mRNAs in Chlamydomonas chloroplasts are formed by 3' end processing of longer precursors.

Animals↗

Relationship between mutation of IR in the mtr system of Neisseria gonorrhoeae and multiple antibiotic resistance.

To study the relationship between mutation of the inverted repeat sequence (IR) in the multiple transferable resistant system (mtr) of Neisseria gonorrhoeae (NG) and its multiple antibiotic resistance, minimal inhibitory concentrations (MICs) for the clinically isolated strains were tested by agar-dilution-method. The mtr system's IR gene of NG was sequenced after amplification by polymerase chain reaction (PCR). Either two susceptive or five penicillin-resistant strains had no base mutation in IR gene, while all of the 13 strains with multiple-antibiotic-resistance had a single-base deletion (A/T). The result suggests that a single-base deletion of the thirteen-base IR sequence in mtr system of NG might result in multiple antibiotic resistance but is not associated with single antibiotic resistance.

Anti-Infective Agents↗

Role of disulfide bond of arylsulfate sulfotransferase in the catalytic activity.

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of sulfate group from a phenyl sulfate ester to a phenolic acceptor. The promoter region and the transcription start sites of Enterobacter amnigenus astA have been determined by primer extension analysis. Northern blot analysis resolved two mRNA species with lengths of 3.3 and 2.0 kb, which correspond to the distances between the transcriptional initiation sites and the two inverted repeat sequences (IRSs). By length, the 3.3 kb RNA could comprise the three-gene (astA with dsbA and dsbB) operon. ASST has three highly conserved cysteine residues. Reducing and nonreducing SDS-PAGE and activity staining showed that disulfide bond is needed for the activity of the enzyme. To identify the cysteine residues responsible for the disulfide bond formation, a series of Cys to Ser mutants has been constructed and the enzymatic activity was measured. Based on the results, we assumed that the first cysteine (Cys349) might be involved in disulfide bond mainly with the second cysteine (Cys445) and result in active conformation.

Arylsulfotransferase↗

Generation of new transposons in vivo: an evolutionary role for the "staggered" head-to-head dimer and one-ended transposition.

From a plasmid carrying the tnpA gene and one inverted repeat sequence (IR) of transposon Tn3, plasmids containing a structure characteristic of transposons, i.e., two IRs flanking a tnpA gene, were generated spontaneously in vivo. They appear to have arisen either through the formation of a "staggered" head-to-head dimer or by so-called one-ended transposition. These putative transposons could indeed transpose to, or form cointegrates with, a recipient plasmid. Based on these findings it is proposed that a primeval transposase gene and its target site evolved first, and subsequently gave rise to a "fully-fledged" transposon by head-to-head dimerization or one-ended transposition.

Base Sequence↗

Distribution of the insertion element IS240 among Bacillus thuringiensis strains.

The presence of IS240 was investigated in 69 Bacillus thuringiensis (Bt) strains including strains from serotype H1 to H45 and additional strains with known Dipteran larvae toxicity. Restriction digests of total DNA and PCR products obtained with a single 16-bases primer corresponding to the IS240 inverted repeated sequence were hybridized with the IS240A element. The results indicate that 67% of the Bt strains tested, including all known mosquitocidal strains, possess at least one IS240-related element. PCR experiments indicate that IS240 represents a family of insertion sequences with several variants.

Animals↗

Evolution of herpes simplex virus type 1 under herpesviral evolutionary processes.

Herpesviruses, the genomes of which are double-stranded DNA of 120 kilobase pairs or more, infect a wide range of vertebrates from mammals to fish. Herpes simplex virus type 1 (HSV-1), a representative of family Herpesviridae, is a ubiquitous human pathogen. HSV-1 relates to common mucocutaneous diseases, while HSV-1 infection can mean a serious outcome, e.g. blindness and insult to the central nervous system. Evolution of herpesviruses includes DNA rearrangements, often generating tandemly or invertedly repeated sequences. Studies of HSV-1 DNA dynamics substantiated these processes of DNA recombination involved in the evolution of herpesvirus. Herpesviruses seem to have diversified from a common ancestor, in a manner mediating co-speciation of herpesviruses with host species through species-specific latent infections. Thus, the notion of host-linked evolution of herpesviruses is given support. Relationships between HSV-1 genotypes and human ethnic groups can be traced by analyses of DNA polymorphisms of HSV-1 strains present in populations of various countries. A close association of an HSV-1 genotype with a particular historical human population seems probable. Such being the case, the host-linked mode is likely to be linked to diversification of HSV-1 in human populations.

Biological Evolution↗

Interactions between structure transitions in a torsionally constrained DNA.

We used S1 nuclease cleavage in conjunction with gel electrophoresis to evaluate torsion-induced cruciform extrusion at two inverted repeat sequences, IRS-B and IRS-C of plasmid pUC12. These structure transitions affect each other through competition for the available torsional free energy according to their relative energies of activation and the magnitude of DNA duplex unwinding associated with each transition. They can be modulated by the level of DNA negative torsion. Interplays between transition sequences occur over long distances and are independent of relative orientation of transition sites. DNA binding factors that enhance or repress structural transitions of specific sequences may, thus, regulate the structural and functional properties of torsionally coupled, distal sequences.

Base Sequence↗

Role of the IS50 R proteins in the promotion and control of Tn5 transposition.

IS50R, the inverted repeat sequence of Tn5 which is responsible for supplying functions that promote and control Tn5 transposition, encodes two polypeptides that differ at their N terminus. Frameshift, in-frame deletion, nonsense, and missense mutations within the N terminus of protein 1 (which is not present in protein 2) were isolated and characterized. The properties of these mutations demonstrate that protein 1 is absolutely required for Tn5 transposition. None of these mutations affected the inhibitory activity of IS50, confirming that protein 2 is sufficient to mediate inhibition of Tn5 transposition. The effects on transposition of increasing the amount of protein 2 (the inhibitor) relative to protein 1 (the transposase) were also analyzed. Relatively large amounts of protein 2 were required to see a significant decrease in the transposition frequency of an element. In addition, varying the co-ordinate synthesis of the IS50 R proteins over a 30-fold range had little effect on the transposition frequency. These studies suggest that neither the wild-type synthesis rate of protein 2 relative to protein 1 nor the amount of synthesis of both IS50 R proteins is the only factor responsible for controlling the transposition frequency of a wild-type Tn5 element in Escherichia coli.

Alleles↗

Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli.

The ilvC gene encodes acetohydroxy acid isomeroreductase (EC 1.1.1.89), the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Expression of the ilvC gene is induced by acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate. This substrate induction is mediated by a positive activator encoded by an adjacent gene, ilvY. The ilvY and ilvC genes are transcribed in opposite directions from promoters that are overlapping. In this paper we characterize the in vitro DNA binding properties of the ilvY-encoded activator protein. The ilvY product binds to two adjacent operator sites located in the divergent-overlapping ilvY and ilvC promoter region. One of these operators, designated O1 contains regions of dyad symmetry centered at position +17 relative to the ilvY transcriptional start site, and the second site, designated O2, contains an homologous inverted repeat sequence centered about the -35 region of the ilvC promoter. Binding of the ilvY product at the O1 and O2 operator sites is co-operative and this ilvY protein-DNA complex in the presence of acetohydroxy acid isomeroreductase substrate is a prerequisite for RNA polymerase binding to the ilvC promoter as detected by DNase I protection experiments. Additionally, chromosomal galK transcriptional fusion assays were performed to characterize the regulation of the ilvY and ilvC promoters in vivo. Transcription of the ilvC gene is maintained at a basal level of activity which is elevated as much as 15-fold in the presence of ilvY product and acetohydroxybutyrate. The ilvY product represses ilvY transcription in a manner that does not appear to be dependent on acetohydroxy acid isomeroreductase substrate. We discuss models in which activation of ilvC transcription results from a direct interaction of ilvY protein with RNA polymerase or an ilvY-mediated alteration of the DNA conformation of the ilvC -35 promoter region. Additionally, we discuss the role of acetohydroxybutyrate and acetolactate in ilvY transcriptional regulation.

2-Acetolactate Mutase↗

Physical characterization of the genome of feline herpesvirus-1.

The physical structure of the genome of feline herpesvirus-1, a major upper respiratory tract pathogen of cats, was studied. Purified FHV-1 DNA was analyzed by restriction endonuclease and gamma 5' exonuclease digestion, blot hybridization, and electron microscopy. To facilitate further studies, nine bacteriophage clones were isolated which contained 85% of the viral genome as SalI inserts, and DNA from these clones was used in blot hybridization experiments and as substrates for restriction digest analysis. Data from these studies permitted construction of a SalI and partial HindII and EcoRI restriction maps of the viral genome. FHV-1 DNA is approximately 134 kb in size and is composed of a long (L) and a short (S) segment. The long segment (U1) is 104 kb in size and is composed of unique DNA. The adjacent S segment is approximately 30 kb in size and contains a central portion of unique DNA (Us) which is approximately 8 kb in size. The Us region is bounded by inverted repeat sequences which are 11 kb in size. Therefore, the physical structure of the FHV-1 genome is similar to the genomes of other alpha-herpesviruses.

Animals↗

Coliphage Q beta RNA replication: RNA catalytic for single-strand release.

We have generated 14 recombinant RNA templates for Q beta replicase, each having either an exogenous inverted repeat sequence or a sequence with no repeat. These templates were used to initiate in vitro replication by Q beta replicase in amounts that saturated the enzyme. We observed that replication rates for RNAs that putatively contained secondary structures in the recombinant sequences ranged from 33 to 69% that of a wild-type MDV-1 RNA control, regardless of the size of the inserted hairpin. Moreover, most of the newly synthesized RNA was present as single strands. Alternatively, RNAs that contained exogenous sequences not expected to form secondary structures exhibited replication rates less than 25% that of MDV-1. In each case, the reaction rate was correlated with the length of the insertion, and the majority of product RNA consisted of duplexed molecules (complementary plus and minus strands hybridized together). When these same recombinant RNAs were used in reactions in which the molar amount of RNA template was 10(6)-10(7) times lower than that of the replicase, only those that putatively contained secondary structures survived in the replication reaction. Our results are consistent with the theory that hairpin structure formation during RNA synthesis by Q beta replicase directly influences the regeneration of single-stranded RNA products.

Base Sequence↗

An electron microscopic study of mouse foldback DNA.

Foldback DNA is defined by its rapid, concentration-independent renaturation, consistent with intramolecular base pairing of inverted repeat sequences. Foldback DNA, isolated from renatured mouse main band DNA by hydroxyapatite chromatography, is spread for electron microscopy by the formamide isodenaturing technique. A large fraction of the molecules can be recognized as intramolecular "hairpins"--structures in which complementary sequences on a single DNA strand form base-paired "stem" regions analogous to tRNA stems. The stem regions of the hairpins have a wide distribution of lengths, averaging about 1000 base pairs. About 60% of the stem regions terminate in single-stranded loops, ranging from 400 to many thousands of nucleotides in length, while 40% of the hairpins do not have discernible loops. There are about 40,000 hairpin-forming sequences in the main band portion of the mouse haploid genome. They appear to be either clustered in groups or confined to about one third of the DNA, rather than uniformly or randomly distributed. Another large fraction of the molecules seen in foldback DNA consists of linear structures, some of which are probably also hairpins. The electron microscopic results, along with simple theoretical considerations, make possible a better interpretation of our previous studies of the yield and S1 nuclease resistance of mouse foldback DNA.

Animals↗

Identification and mapping of the transcriptional and translational products of the yeast plasmid, 2mu circle.

We have identified two major and approximately ten minor poly(A)-containing RNA species in S. cerevisiae which arise from in vivo transcription of the yeast plasmid, known as 2mu circle. The two major species, which are 1325 and 1275 bases in length, are transcribed from the two unique halves of the plasmid and extend into the inverted repeat sequences which separate the unique regions. The map positions of the minor transcripts, which range in length from 350 to 2600 bases, indicate that except for a small region of the genome in which no transcription is observed, both strands of the entire 2mu circle genome are transcribed. We also present evidence demonstrating that RNA transcribed from 2mu circular DNA is used to program the synthesis of specific proteins in yeast: that is, yeast RNA complementary to 2mu circle DNA can be translated in vitro to produce specific polypeptides of substantial size. Finally, the pattern of transcription of 2mu circle suggests the possibility that messenger RNA species are derived by cleavage of larger transcripts, and in addition, that the intramolecular recombination of 2mu circle which occurs in yeast functions as a genetic switch to allow separate expression of two sets of genes on the 2mu circle genome.

Binding Sites↗