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PubMed1985

Post-transcriptional regulation of chloramphenicol acetyl transferase.

The +1 site for initiation of inducible chloramphenicol acetyl transferase (CAT) messenger ribonucleic acid (mRNA) encoded by plasmid pC194 was determined experimentally using gamma-32P-ATP-labeled run-off transcripts partially digested with T1 ribonuclease. By partial digestion of the in vitro transcripts with S1-, T1-, and cobra venom nucleases as probes of mRNA conformation, single- and double-stranded regions, respectively, were also identified. Thus, a prominent inverted complementary repeat sequence was demonstrated spanning the +14 to +50 positions which contain the complementary sequences CCUCC, and GGAGG (the Shine and Dalgarno sequence for synthesis of CAT) symmetrically apposed and paired as part of a perfect 12 bp inverted complementary repeat sequence (-19.5 kcal/mol). The CAT mRNA was stable to digestion by T1 ribonuclease at the 4 guanosine residues in the Shine and Dalgarno sequence GGAGG, even at 60 degrees C, suggesting that nascent CAT mRNA allows ribosomes to initiate protein synthesis inefficiently and that induction involves post-transcriptional unmasking of the Shine and Dalgarno sequence. Consistent with this model of regulation, we found that cells carrying pC194, induced with chloramphenicol (CAM), contain about the same concentration of pulse labeled CAT-specific RNA as do uninduced cells. Induction of CAT synthesis by the nonacetylatable CAM analog fluorothiamphenicol was tested using minicells of Bacillus subtilis carrying pC194 as well as minicells containing the cloned pC194 derivatives in which parts of the CAT structural gene were deleted in vitro using Ba131 exonuclease. Optimal induction of both full-length (active) and deleted (inactive) CAT required similar concentrations of fluorothiamphenicol, whereas induction by CAM required a higher concentration for the wild-type full-length (active) CAT than for the (inactive) deleted CAT. Because synthesis of deleted CAT was inducible, we infer that CAT plays no direct role in regulating its own synthesis.

Acetyltransferases↗
PubMed2004

Promoter activity of left inverted terminal repeat and downstream sequences of porcine adenovirus type 3.

Early region 1 (E1) of porcine adenovirus type 3 (PAdV-3) consists of E1A and E1B transcription units. The authentic promoter region of E1A contains a TATA box at nucleotide position (nt) 449 and a bifunctional regulatory element between nt 374 and 431, which enhances the transcription of E1A, but represses that of E1B. Here, we investigated the role of the left inverted terminal repeat (ITR) and its downstream sequences (between nt 151 and 312) in the transcription of early viral genes, and viral replication. Mutant PAdV-3s without the authentic E1A promoter region could be rescued by transfection of mutant genomic DNA into fetal porcine retina cells. Moreover, the mutant PAdV-3s produced E1A-specific mRNA and remained viable in swine testis (ST) cells suggesting that the left-terminal 151 bp including the ITR, can serve as a promoter for E1A expression. However, mutant PAdV-3s containing deletion including authentic E1A promoter region, displayed both reduced steady-state levels of early gene mRNAs (E1A, E1B, E2A, E3, and E4) and decreased rate of viral replication in ST cells. Interestingly, mutant PAdV-3s containing the left-terminal 312 bp displayed increased transcription of early genes including E1A. Our results suggest that the left ITR of PAdV-3 contain the promoter like elements and the sequences (between nt 151 and 312) downstream of left ITR can enhance its promoter activity.

Adenovirus E1A Proteins↗
PubMed1984

Post-transcriptional regulation of chloramphenicol acetyl transferase.

The +1 site for initiation of inducible chloramphenicol acetyl transferase (CAT) mRNA encoded by plasmid pC194 was determined experimentally by using [alpha-32P]ATP-labeled runoff transcripts partially digested with T1 RNase. By partial digestion of the in vitro transcripts with S1, T1, and cobra venom nucleases as probes of mRNA conformation, single- and double-stranded regions, respectively, were also identified. Thus, a prominent inverted complementary repeat sequence was demonstrated spanning the +14 to +50 positions, which contain the complementary sequences CCUCC and GGAGG (the Shine and Dalgarno sequence for synthesis of CAT) symmetrically apposed and paired as part of a perfect 12-base-pair inverted complementary repeat sequence (-19.5 kcal [ca. -81.7 kJ] per mol). The CAT mRNA was stable to digestion by T1 RNase at the four guanosine residues in the Shine and Dalgarno sequence GGAGG , even at 60 degrees C, suggesting that nascent CAT mRNA allows ribosomes to initiate protein synthesis inefficiently and that induction involves post-transcriptional unmasking of the Shine and Dalgarno sequence. Consistent with this model of regulation, we found that cells carrying pC194 , induced with chloramphenicol, contain about the same concentration of pulse-labeled CAT-specific RNA as do uninduced cells. Induction of CAT synthesis by the non- acetylatable chloramphenicol analog fluorothiamphenicol was tested by using minicells of Bacillus subtilis carrying pC194 as well as minicells containing the cloned pC194 derivatives in which parts of the CAT structural gene were deleted in vitro with BAL 31 exonuclease. Optimal induction of both full-length (active) and deleted (inactive) CAT required similar concentrations of fluorothiamphenicol, whereas induction by chloramphenicol required a higher concentration for the wild-type full-length (active) CAT than for the (inactive) deleted CAT. Because synthesis of deleted CAT was inducible, we infer that CAT plays no direct role in regulating its own synthesis.

Acetyltransferases↗
PubMed1986

Conversion of a fraction of the unique sequence to part of the inverted repeats in the S component of the herpes simplex virus type 1 genome.

A novel genome variant of herpes simplex virus type 1 (HSV-1) was isolated, in the S component of which a fraction of the unique sequence (map units 0.865 to 0.880) was converted to part of the diploid inverted repeats and another fraction of the unique sequence (map units 0.937 to 0.955) had been deleted. The S component of the variant consisted of a shortened unique sequence (map units 0.880 to 0.937) and a pair of elongated inverted repeats (map units 0.820 to 0.880, and 0.937 to 1.000). The conversion occurred as a result of a recombination event between two points (map units 0.880 and 0.937) having a 5 base pair stretch (5'-CCCCG-3') of homology, in an inverted direction in the unique sequence. An involvement of the mechanism causing L-S inversion was inferred to account for the generation of the variant, as there are multiple copies of the 5'-CCCCG-3' stretch in the 'a' sequences. The occurrence of the variant indicates that the products of HSV-1 genes US9, US10, US11 and US12 are unnecessary for an HSV-1 productive infection in tissue culture cells, and also suggests the presence of a mechanism whereby expansion of the inverted repeats could occur.

Base Sequence↗
PubMed1994

Regulation of plasmid pE194 replication: control of cop-repF operon transcription by Cop and of repF translation by countertranscript RNA.

The cop-rep region of plasmid pE194 contains two tandem structural genes, cop and repF, as well as the plus and minus origins of replication. The two structural genes comprise an operon whose expression is repressed by the binding of Cop protein to a 28-bp inverted complementary repeat sequence that overlaps the cop-repF promoter. From its position relative to the promoter and the experimentally determined footprint made by the Cop protein, the 28-bp inverted complementary repeat sequence is presumed to function as the cop operator. The intercistronic region between cop and repF is 80 nucleotides (nt) long and is transcribed bidirectionally: in the forward direction as part of the synthesis of the cop-repF message (ca. 900 nt), and in the reverse direction to yield a countertranscript ca. 65 nt long. The proposed countertranscript RNA (ctRNA) can form a single stem-and-loop structure that includes the single SphI sequence of plasmid pE194 as part of the loop-forming segment. Enlargement of the proposed loop from 6 to 14 nt by insertion of a SphI-BamHI adapter at the SphI site or contraction of the proposed loop down to 4 nt, by cutting with SphI followed by blunting with S1 nuclease, yields mutants with an increased copy number. By gel retardation and DNaseI footprinting analysis, Cop protein was shown to bind to the promoter region of cop; no binding by Cop protein at the 5' end of repF was detected. Two major transcripts were synthesized in vitro by using cop-repF region DNA as a template, the tandem cop-repF transcript, and the ctRNA. Addition of purified Cop protein to an vitro transcription reaction mixture reduced only the rate of cop-repF transcription but not that of ctRNA. These observations suggest that regulations of repF occurs at two levels: (i) with Cop protein acting as a repressor of cop-repF mRNA transcription and (ii) with ctRNA acting as a repressor of RepF translation.

Amino Acid Sequence↗
PubMed1990

Evolution of the human alpha-amylase multigene family through unequal, homologous, and inter- and intrachromosomal crossovers.

Human amylase haplotypes differ from each other by different numbers of a long direct repeat unit of approximately 100 kb, encompassing two complete salivary amylase genes and one amylase pseudogene lacking the first three exons. The two salivary genes are part of a 75-kb-long inverted repeat. Two short sequences, hybridizing with a probe containing exons 1-3, were found in the central part of the inverted repeat. Sequencing showed that these fragments, designated r, contain exon 3 sequences. We present evidence that these r-fragments and the pseudogene most likely are remnants of the same ancestral pancreatic gene. We determined the orientation of the exon 3 sequences present in the r-fragment and show that an inversion can explain their origination. Hybridization studies, using random fragments from the intergenic region of the AMY gene cluster as probes, enabled us to detect more extended homologous regions in this cluster than were found previously on the basis of restriction maps only. Together, these results allow us to present a model for the evolution of the human amylase multigene family by a number of consecutive events involving inter- and intrachromosomal crossovers.

Base Sequence↗
PubMed1978

Sequence organization of the rat genome by electron microscopy.

The size and arrangement of repetitive and inverted repeat (foldback) sequences in rat DNA were studied by visualization of hybrid and heteroduplex structures in the electron microscope. The self-reassociation of repetitive sequence-bearing DNA strands often results in the formation of four-ended "H" structures, whose duplex regions equal the repetitive sequence length and can be measured in the electron microscope. In this way, it was determined that the average size of the class of numerous short repetitive sequences is 0.40 +/- 0.15 kbp. Heteroduplex structures were prepared between long whole DNA single strands and short repeat-sequence-bearing strands. The analysis of these structures confirms that the size of the repetitive sequences in 0.4 kbp on average. Length measurements between adjacent duplexes show that the average spacing between two interspersed repeats is at least 1.5-1.8 kbp. By examining 29.4-kbp single strands after brief renaturation, the size and distribution of foldback sequences were determined. There are 1.9 X 10(5) foldback apirs per rat genome, spaced an average of 9.7 kbp apart according to our measurement. Repetitive, inverted repeat and unique sequences are interspersed with each other in at least half the genome.

Animals↗
PubMed1995

Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes.

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.

Base Sequence↗
PubMed1993

Functional in vivo analyses of the 3' flanking sequences of the Chlamydomonas chloroplast rbcL and psaB genes.

Possible roles of untranslated sequences at the 3' ends of chloroplast genes, which include inverted repeat elements, were investigated in Chlamydomonas reinhardtii in vivo. Chlamydomonas chloroplast rbcL or psaB 3' flanking regions were coupled in various arrangements 3' to a chimeric gene consisting of a Chlamydomonas chloroplast atpB promoter sequence fused 5' to the Escherichia coli uidA (GUS) structural gene. These genes were introduced into the Chlamydomonas chloroplast genome at the same location by homologous recombination following microprojectile bombardment. Transformants harboring chimeric GUS genes fused to rbcL or psaB gene 3' inverted repeat sequences in their normal forward orientations accumulated GUS transcripts of a single size, whereas GUS transcripts of heterogenous sizes accumulated in transformants harboring the same gene lacking an inverted repeat sequence at its 3' end. Thus, the 3' flanking regions of the rbcL and psaB genes can define the location of the 3' terminus of a transcript in vivo. In chloroplast transformants harboring chimeric GUS genes fused to multiple inverted repeat sequences in their normal forward orientations, only GUS transcripts accumulated that were terminated by the first inverted repeat sequence. The latter data suggest that the 3' ends of these RNAs are the products of either transcription termination or endonucleolytic cleavage. Analyses of GUS transcripts in transformants harboring GUS genes terminated by rbcL or psaB gene 3' flanking regions in reversed orientations indicate that transcript 3' end formation in vivo requires nucleotide sequences located outside the inverted repeat elements. Inasmuch as decay rates of GUS transcripts were found to be independent of the presence of a 3' inverted repeat sequence, RNA stabilization does not appear to be a major in vivo function of these elements in the Chlamydomonas chloroplast transcripts studied.

Animals↗
PubMed1993

Instability of a plasmid-borne inverted repeat in Saccharomyces cerevisiae.

Inverted repeated DNA sequences are common in both prokaryotes and eukaryotes. We found that a plasmid-borne 94 base-pair inverted repeat (a perfect palindrome of 47 bp) containing a poly GT sequence is unstable in S. cerevisiae, with a minimal deletion frequency of about 10(-4)/mitotic division. Ten independent deletions had identical end points. Sequence analysis indicated that all deletions were the result of a DNA polymerase slippage event (or a recombination event) involving a 5-bp repeat (5' CGACG 3') that flanked the inverted repeat. The deletion rate and the types of deletions were unaffected by the rad52 mutation. Strains with the pms1 mutation had a 10-fold elevated frequency of instability of the inverted repeat. The types of sequence alterations observed in the pms1 background, however, were different than those seen in either the wild-type or rad52 genetic backgrounds.

Base Sequence↗
PubMed1991

Analysis of inverted repeats in primary structure of proteins.

A computer program has been developed to locate exact inverted repeating subsequences present anywhere in the given primary structure of proteins or nucleic acids. The output is amenable to protein sequence/nucleic acid query (PSQ/NAQ) packages. Our analysis has shown that there is a large number of proteins which have inverted repeats of more than four amino acid residues in length. However, the number is small when conditions such as the existence of more than 20 inverted repeats in given sequence or the existence of inverted repeats having more than five different types of amino acids are applied.

Amino Acid Sequence↗
PubMed1984

Replication of adenovirus mini-chromosomes.

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear mini-chromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.

Adenoviridae↗
PubMed1993

[Nucleotide sequence and properties of an inverted repeating element of a Bordetella pertussis chromosome].

The repeated sequence from Bordetella pertussis chromosome was cloned using the method described by Ohtsubo. The sequences of the characterized B. pertussis chromosome are homologous to the previously described sequences and analogous in the structure to the already known IS elements. The parental recombinant plasmids containing the RS were unstable and segregated to the plasmids of different structure. The segregants' structure is characterized in this paper. It is shown in our study that the RS element is able to stimulate intragenomic rearrangements, such as deletions. It is shown that at least one deletion begins precisely after 3' end of RS and terminates with the sequence which is completely homologous to ten terminal nucleotides of this one. Probably, RSs stimulate at high frequency the formation of deletions, which appear to be the result of recA-independent site-specific recombination between short direct repeats.

Base Sequence↗
PubMed1982

Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance.

The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, has been determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, have been mapped functionally by subcloning a 1,035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1,035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinant cloned in pBR322 could not replicate in B. subtilis, ability to replicate in B. subtilis was seen if the fragment cloned included an extension consisting of an additional 300 nucleotides beyond the 5' end of the single pC194 MspI site associated with replication. This 5' extension contained a 120-nucleotide inverted complementary repeat sequence similar to that found in pE194 TaqI fragment B which contains replication sequences of that plasmid. pC194 was found to contain four opening reading frames theoretically capable of coding for proteins with maximum molecular masses, as follows: A, 27,800 daltons; B, 26,200 daltons; C, 15,000 daltons; and D, 9,600 daltons. Interruption or deletion of either frame A or D does not entail loss of ability to replicate or to express CAM resistance, whereas frame B contains the CAT structural gene and frame C contains sequences associated with plasmid replication.

Acetyltransferases↗
PubMed1987

The effects of deletions in the leader sequence of cat-86, a chloramphenicol-resistance gene isolated from Bacillus pumilus.

The cat-86 gene of Bacillus pumilus, specifying a Cm-inducible CAT enzyme, was cloned previously into B. subtilis on plasmid pUB110. Various lines of evidence suggest that control of expression of this gene is at the level of translation and involves inverted complementary repeat sequences 5' to the initiation codon. A series of deletions have been generated in this region and their effects on the induction of cat-86 observed in B. subtilis, Escherichia coli and a number of ribosomal mutant strains of B. subtilis. The results indicate that the inverted complementary repeat sequences, which are capable of forming a stable stem-loop structure in the mRNA (delta G = -24.4 kcal/mol), form a barrier to translation in E. coli and B. subtilis.

Acetyltransferases↗
PubMed1986

Conservation of sequences adjacent to the telomeric C4A2 repeats of ciliate macronuclear ribosomal RNA gene molecules.

We sequenced and compared the telomeric regions of linear rDNAs from vegetative macronuclei of several ciliates in the suborder Tetrahymenina. All telomeres consisted of tandemly repeated C4A2 sequences, including the 5' telomere of the 11 kb rDNA from developing macronuclei of Tetrahymena thermophila. Our sequence of the 11 kb 5' telomeric region shows that each one of a previously described pair of inverted repeats flanking the micronuclear rDNA (Yao et al., Mol. Cell. Biol. 5: 1260-1267, 1985) is 29 bp away from the positions to which telomeric C4A2 repeats are joined to the ends of excised 11 kb rDNA. In general we found that the macronuclear rDNA sequences adjacent to C4A2 repeats are not highly conserved. However, in the non-palindromic rDNA of Glaucoma, we identified a single copy of a conserved sequence, repeated in inverted orientation in Tetrahymena spp., which all form palindromic rDNAs. We propose that this sequence is required for a step in rDNA excision common to both Tetrahymena and Glaucoma.

Animals↗
PubMed1999

A mutation in the flanking 5'-TA-3' dinucleotide prevents excision of an internal eliminated sequence from the Paramecium tetraurelia genome.

The germline chromosomes in Paramecium and other ciliated protozoa contain regions of DNA that are excised and eliminated during the development of a new macronuclear genome. Paramecium tetraurelia internal eliminated sequences (IESs) are invariably flanked by a 5'-TA-3' dinucleotide sequence that is part of a larger 8-bp terminal inverted-repeat consensus sequence. Both features, the absolutely conserved 5'-TA-3' and the remaining 6-bp terminal inverted repeat, are shared with the mariner/Tc1 class of transposons. In this article we describe a mutant cell line (AIM-2) defective in excision of a single IES from the coding region of the A51 surface antigen gene. Excision of the 370-bp IES6649 is prevented by a single A to G transition in the invariably conserved 5'-TA-3' dinucleotide. Failure to excise IES6649 also revealed a 29-bp IES located inside IES6649. Additional experiments with the previously isolated AIM-1 mutant, which also contains an internal IES, shows that alternate excision using the wild-type end of IES2591 with an end from the internal IES is extremely rare or nonexistent. These results indicate that IESs are discrete elements whose excision depends upon nucleotides located within the consensus sequence, but also suggest that additional information is required to match one end of an IES with its excision partner.

Animals↗
PubMed1988

Sequence identity between an inverted repeat family of transposable elements in Drosophila and Caenorhabditis.

The Tc1-like transposable elements, originally described in Caenorhabditis elegans, have a much wider phylogenetic distribution than previously thought. In this paper, we demonstrate that Tc1 shares sequence identity in its open reading frame and terminal repeats with a new transposable element Barney (also known as TCb1-Transposon Caenorhabditis briggsae 1). Barney was detected and isolated by Tc1 hybridization from the closely related nematode species, Caenorhabditis briggsae. The conserved open reading frames of Tc1 and Barney share identity with a structurally similar family of elements named HB found in Drosophila melanogaster, after the introduction of 3 small centrally located deletions in HB1. These reading frames would code for proteins with 30% amino acid identity (42% when conservative changes are included). Tc1, Barney and HB1 contain highly conserved blocks of amino acids which are likely to be in the functional domains of the putative transposase.

Amino Acid Sequence↗