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Structural analysis of the fibroin gene at the 5' end and its surrounding regions.

Using the genomic clones of the fibroin gene with its flanking sequences, a detailed restriction map was prepared with particular attention given to the region neighboring the 5' end of the gene. About 60% of the fibroin mRNA has a complete cap (m7GpppAmUmCXG), and the remaining 40% has an unmethylated cap (GpppAPyXXG). The latter can be radioactively labeled by the use of vaccinia capping enzymes. Using the labeled mRNA, we mapped the 5' end of the gene by hybridization to restriction enzyme digests. DNA sequencing of this region revealed a sequence ATCAGCATCAG that corresponds to ApyXXG in tandem. The 5' end of mRNA with a complete cap was also located in these 11 nucleotides. Since the second ATCAG region was protected from S1 digestion following hybridization with total fibroin mRNA, we have tentatively assigned it as the sole 5' end of the transcription unit. About 700 bp were sequenced in the region from -800 + 1700 bp. The region of about 210 bp preceding the 5' end of the gene which may accommodate a promoter sequence is very AT-rich and contains clusters of A and T residues. This region contains statistically significant dyad symmetries, direct repeats and inverted repeats. At -30 to -24, the sequence TATAAAA was observed, and this is similar to a Pribnow-type sequence (TATAGATG). The sequence that codes for the gly-ala repetitious peptide characteristic of fibroin begins somewhere between 1340 and 1600 bp from the 5' end. An intervening sequence of about 1.1 kb that interrupts the gene about 70 bp from the 5' end was detected by R loop formation. Structures of the fibroin gene cloned from fibroin producing and nonproducing cells look the same, especially around the 5' end and the intervening region.

Animals↗

An extensively edited mitochondrial transcript in kinetoplastids encodes a protein homologous to ATPase subunit 6.

The mitochondrial MURF4 gene of T. brucei has pronounced G versus C strand bias and heterogeneously sized transcripts, characteristic of genes encoding extensively edited transcripts. We find that MURF4 transcripts of T. brucei are extensively edited throughout by the addition and deletion of numerous uridines, creating potential initiation and termination codons and a continuous open reading frame. A potential guide RNA sequence occurs in a minicircle between inverted repeats. The 5' region of L. tarentolae MURF4 transcripts is also extensively edited, with a created initiation codon. The predicted MURF4 amino acid sequences have homology to those of mitochondrial ATP-ase subunit 6 genes from a variety of organisms. In addition, their hydropathic profiles are quite similar to those of other species. We therefore conclude that MURF4 encodes ATPase subunit 6 genes.

Adenosine Triphosphatases↗

Diversity of genetic environment of bla(CTX-M) genes.

Twenty-four non-clonally related enterobacterial isolates producing the emerging CTX-M-type extended-spectrum beta-lactamases were recovered from several countries including France, India, Poland, and Turkey. They had been isolated from 2000 to 2003. beta-Lactamases CTX-M-2, CTX-M-3, CTX-M-10, CTX-M-14 and CTX-M-15 were identified. Most of the isolates produced beta-lactamase CTX-M-15. Insertion sequence ISEcp1 was found upstream of bla(CTX-M-3), bla(CTX-M-10), bla(CTX-M-14) and bla(CTX-M-15) genes. A sequence similar to the inverted right repeat of ISEcp1 was identified downstream of bla(CTX-M-3), bla(CTX-M-10) and bla(CTX-M-15) genes suggesting the mobilization of these beta-lactamase genes by transposition events. In addition, Orf513 was identified upstream of the bla(CTX-M-2) gene. This work further underlined widespread of bla(CTX-M-15) gene associated with ISEcp1.

Base Sequence↗

A gene cluster for chlorate metabolism in Ideonella dechloratans.

Chlorate reductase has been isolated from the chlorate-respiring bacterium Ideonella dechloratans, and the genes encoding the enzyme have been sequenced. The enzyme is composed of three different subunits and contains molybdopterin, iron, probably in iron-sulfur clusters, and heme b. The genes (clr) encoding chlorate reductase are arranged as clrABDC, where clrA, clrB, and clrC encode the subunits and clrD encodes a specific chaperone. Judging from the subunit composition, cofactor content, and sequence comparisons, chlorate reductase belongs to class II of the dimethyl sulfoxide reductase family. The clr genes are preceded by a novel insertion sequence (transposase gene surrounded by inverted repeats), denoted ISIde1. Further upstream, we find the previously characterized gene for chlorite dismutase (cld), oriented in the opposite direction. Chlorate metabolism in I. dechloratans starts with the reduction of chlorate, which is followed by the decomposition of the resulting chlorite to chloride and molecular oxygen. The present work reveals that the genes encoding the enzymes catalyzing both these reactions are in close proximity.

Amino Acid Sequence↗

A physical and functional analysis of Tn917, a Streptococcus transposon in the Tn3 family that functions in Bacillus.

The erythromycin-resistance (Emr)-conferring transposon Tn917, first isolated in the genus Streptococcus, has in previous work been shown to function efficiently in the spore-forming species Bacillus subtilis, where it has been developed as a tool for identifying and studying sporulation genes. In the present work, a physical analysis of Tn917 was undertaken, including detailed restriction mapping, chemical DNA sequencing, heteroduplex studies, and Southern hybridization analysis, as a first step in understanding the genetic organization of this useful insertion element. The location and transcriptional orientation of the transposon-borne erm gene (the gene responsible for the Emr phenotype) have been determined, and a partial sequence of DNA 5' to the coding sequence of this gene indicates that its inducibility is probably the result of "translational attenuation," a mechanism known to be responsible for the regulation of at least two other gram-positive erm genes. Restriction mapping and heteroduplex analysis have revealed extensive homology between Tn917 and the Staphylococcus transposon Tn551, throughout virtually their entire lengths, and DNA sequencing studies have revealed a remarkably high degree of sequence correspondence within the terminal inverted repeats of Tn917, Tn551 and the gram-negative transposon Tn3. Tn917 was also shown to generate a 5-bp duplication upon insertion, as do Tn3 and Tn551 (and all of the other Tn3-related elements studied thus far), strengthening the conclusion that these three transposons are members of a highly dispersed family of related insertion elements which populate both gram-positive and gram-negative genera.

Bacillus↗

Nucleotide sequence analysis of IS427 and its target sites in Agrobacterium tumefaciens T37.

We have determined the nucleotide sequence of IS427, an insertion sequence from Agrobacterium tumefaciens T37, IS427 is 1271 bp long, contains 16-bp imperfect terminal inverted repeats, and generates a 2-bp target sequence duplication. It is present at three sites in the pTiT37 plasmid and is absent from the chromosome of A. tumefaciens T37. Each of the IS427 elements sequenced was near a site with sequence homology to integration host factor (IHF)-binding sites which suggested that IHF may be involved in IS427 transposition.

Amino Acid Sequence↗

Multigene families in African swine fever virus: family 360.

A group of cross-hybridizing DNA segments contained within the restriction fragments RK', RL, RJ, and RD' of African swine fever virus DNA were mapped and sequenced. Analysis of these sequences revealed the presence of a family of homologous open reading frames in regions close to the DNA ends. The whole family is composed of six open reading frames with an average length of 360 coding triplets (multigene family 360), four of which are located in the left part of the genome and two of which are in the right terminal EcoRI fragment. In close proximity to the right terminal inverted repeat, we found an additional small open reading frame which was homologous to the 5'-terminal portion of the other open reading frames, suggesting that most of that open reading frame has been deleted. These repeated sequences account for the previously described inverted internal repetitions (J.M. Sogo, J.M. Almendral, A. Talavera, and E. Viñuela, Virology 133:271-275, 1984). Most of the genes of multigene family 360 are transcribed in African swine fever virus-infected cells. A comparison of the predicted protein sequences of family 360 indicated that several residues are conserved, suggesting that an overall structure is maintained for every member of the family. The transcription direction of each open reading frame, as well as the evolutionary relationships among the genes, suggests that the family originated by gene duplication and translocation of sequences between the DNA ends.

Amino Acid Sequence↗

A variant Tc4 transposable element in the nematode C. elegans could encode a novel protein.

A variant C. elegans Tc4 transposable element, Tc4-rh1030, has been sequenced and is 3483 bp long. The Tc4 element that had been analyzed previously is 1605 bp long, consists of two 774-bp nearly perfect inverted terminal repeats connected by a 57-bp loop, and lacks significant open reading frames. In Tc4-rh1030, by comparison, a 2343-bp novel sequence is present in place of a 477-bp segment in one of the inverted repeats. The novel sequence of Tc4-rh1030 is present about five times per haploid genome and is invariably associated with Tc4 elements; we have used the designation Tc4v to denote this variant subfamily of Tc4 elements. Sequence analysis of three cDNA clones suggests that a Tc4v element contains at least five exons that could encode a novel basic protein of 537 amino acid residues. On northern blots, a 1.6-kb Tc4v-specific transcript was detected in the mutator strain TR679 but not in the wild-type strain N2; Tc4 elements are known to transpose in TR679 but appear to be quiescent in N2. We have analyzed transcripts produced by an unc-33 gene that has the Tc4-rh1030 insertional mutation in its transcribed region; all or almost all of the Tc4v sequence is frequently spliced out of the mutant unc-33 transcripts, sometimes by means of non-consensus splice acceptor sites.

Amino Acid Sequence↗

E1 recognition sequences in the bovine papillomavirus type 1 origin of DNA replication: interaction between half sites of the inverted repeats.

The E1 protein encoded by bovine papillomavirus type 1 (BPV-1) is required for viral DNA replication, and it binds site specifically to an A/T-rich palindromic sequence within the viral origin of replication. The protein is targeted to this site through cooperative interactions and binding with the virus-encoded E2 protein. To explore the nature of the E1 binding site, we inserted a series of homologous DNA linkers at the center of dyad symmetry within the E1 recognition palindrome. The effects of these modifications indicated that the E1 recognition palindrome can be separated into functional half sites. The series of insertions manifest a phasing relationship with respect to the wild-type BPV-1 genome in that greater biological activity was measured when full integral turns of the DNA helix separated the palindrome than when the separations were half-turns. This phasing pattern of activity was observed to occur in a variety of biological phenotypes, including transformation efficiency, stable plasmid copy number in cell lines established from pooled foci, and transient replication of full-length viral genomes. For replication reporter constructs where E1 and E2 are supplied in trans by the respective expression vectors, distance between the half sites seems to play a major role, yet the phasing relationships are measurable. DNase I protection studies showed that E1 bound very poorly to the construct containing a 5-bp linker, and binding was close to the wild-type level for the 10-bp insertion, consistent with a requirement for a phasing function between half sites with a modulus of 10 bp. Binding to the 15- and 20-bp insertion mutants was weak, but only for the 20-bp insertions was protection over both halves of the palindrome measurable. As it had been previously reported that the 18-bp palindrome contains sufficient nucleotide sequence information for E1 binding, we speculate that a minimal E1 recognition motif is presented in each half site. A comparison between this sequence and that of an upstream region that also binds E1 (the E2RE1 region) revealed a common pentanucleotide motif of APyAAPy. Mutants with substitutions of the ATAAT elements within E2RE1 failed to bind E1 protein. We present models for how repeats of the pentanucleotide sequence may coordinate E1 binding at the dyad symmetry axis of the origin and compare the DNA sequence organization of BPV-1 with those of the simian virus 40 and polyomaviruses at their origins of DNA replication.

Base Sequence↗

Selective DNA binding by the androgen receptor as a mechanism for hormone-specific gene regulation.

Steroid hormones control many physiological processes by activating specific receptors that act as transcription factors. In vivo, each of these receptors has a specific set of target genes, but in vitro the glucocorticoid, progesterone, mineralocorticoid and androgen receptors (class I receptors) all recognise response elements which are organised as inverted repeats of 5'-TGTTCT-3'-like sequences with a three nucleotide spacer. This poses the question how the in vivo specificity of the different steroid responses is mediated. To unravel the mechanisms involved, we have compared the structural features of the androgen-selective enhancers of the probasin, the secretory component and the sex-limited protein genes with those of non-selective enhancers in the mouse mammary tumour viral promoter and the C3(1) gene. The probasin promoter contains an androgen response element which is recognised with high affinity by the androgen receptor, but not by the other class I receptors. Swapping experiments between the DNA-binding domains of the androgen and glucocorticoid receptor revealed that it is not the first zinc finger, but rather the second zinc finger and part of the hinge region which contribute to this specificity. Three AR-specific aminoacids are involved in the probasin ARE recognition, but not in the C3(1) ARE binding by the AR. The location of these residues strongly suggests that an alternative dimerisation interface is involved in the probasin ARE binding. We could subsequently demonstrate that the AR binds direct repeats of 5'-TGTTCT-3'-like sequences in gel retardation assays as well as in transfection experiments. Moreover, the androgen-specific enhancers all contain direct repeats, and point mutations that change the nature of these elements into inverted repeats result in a change of specificity. It seems, therefore, that direct repeat elements can be the determinants of the AR-specificity. It will be exciting to learn how such DNA elements will affect the properties of the receptor dimer with respect to ligand binding, interactions between the aminoterminal domain and the ligand-binding domain, the recruitement of co-activators and cooperativity with other transcription factors.

Animals↗

Riboflavin operon of Bacillus subtilis: unusual symmetric arrangement of the regulatory region.

Seventeen cis-dominant mutations leading to riboflavin overproduction in Bacillus subtilis were localized to the region between nucleotides +37 and +159 relative to the transcription initiation site of the riboflavin operon. This region displays an unusual structure for regulatory sequences. The main part of it represents clusters of A/T and G/C-rich sequences that symmetrically blank a short inverted repeat.

Bacillus subtilis↗

A Tn21 terminal sequence within Tn501: complementation of tnpA gene function and transposon evolution.

The prokaryotic mercury-resistance transposon Tn501 contains a sequence, 80 nucleotides from one end, which is identical with an inverted terminal repeat (IR) of Tn21. This Tn21 IR sequence is used when Tn21 complements a TnpA- derivative of Tn501, but not when Tn501 is used for the complementation. Complementation by Tn1721 shows a preference for the normal Tn501 IRs. The element (Tn820) transposed when Tn21 is used to complement a Hg- TnpR- TnpA- Res- deletion mutant of Tn501 contains the Tn21 IR sequence at one terminus and a Tn501 IR at the other. Transposition of Tn820 can be complemented by Tn501 and Tn1721, but at a much lower frequency than transposition of the parental element (Tn819) which has two Tn501 IRs. The relationship between the transposition functions of Tn501, Tn21 and Tn1721, and available nucleotide sequence data suggest that Tn501 evolved by the transposition of a Tn21-like element into another transposable element (similar to that found within Tn1721) followed by deletion of the Tn21-like transposition functions.

Base Sequence↗

Binding sites for maize nuclear proteins in the subterminal regions of the transposable element Activator.

Genetic data suggest that transposition of the maize element Activator (Ac) is modulated by host factors. Using gel retardation and DNase I protection assays we identified maize proteins which bind to seven subterminal sites in both ends of Ac. Four DNase I-protected sites contain a GGTAAA sequence, the other three include either GATAAA or GTTAAA. The specificity of the maize protein binding to Ac was verified by using a synthetic fragment containing four GGTAAA motifs as probe and competitor in gel retardation assays. All seven binding sites are located within regions required in cis for transposition. A maize protein binding site with the same sequence has previously been identified in the terminal inverted repeats of the maize Mutator element. Thus, the protein, that recognizes this sequence is a good candidate for a regulatory host factor for Ac transposition.

Base Sequence↗

Specific sequences and a hairpin structure in the template strand are required for N4 virion RNA polymerase promoter recognition.

Coliphage N4 virion-encapsidated, DNA-dependent RNA polymerase (vRNAP) is inactive on double-stranded N4 DNA; however, denatured promoter-containing templates are accurately transcribed. We report that all determinants of vRNAP promoter recognition exist in the template strand, indicating that this enzyme is a site-specific, single-stranded DNA-binding protein. We show that conserved sequences and the integrity of inverted repeats present at the promoters are essential for activity, suggesting the necessity for specific secondary structure. Evidence for such a structure is presented. We propose a model for in vivo utilization of vRNAP promoters in which template negative supercoiling yields single-strandedness at the promoter to reveal the determinants of vRNAP binding. This structure is stabilized by the binding of E. coli single-stranded DNA-binding protein to yield an "activated promoter."

Affinity Labels↗

Analysis of hypermethylation in the RPS element suggests a signal function for short inverted repeats in de novo methylation.

A repetitive DNA sequence (RPS) from Petunia hybrida had previously been shown to enhance expression variegation in petunia and tobacco and to carry a hot spot for de novo DNA methylation. Here we show that a strong de novo hypermethylation site is located within a palindromic segment of the RPS and present indirect evidence, based on sequence homologies to other repeat units within the RPS, for the formation of secondary structures at the methylation site in vivo. We demonstrate that the palindromic RPS element, which is moderately to highly repetitive in petunia, does not predominantly localise to constitutive heterochromatin. To test whether the RPS is subject to de novo methylation due to its repetitive nature or to intrinsic signals within the RPS, we integrated the RPS into the genome of Arabidopsis thaliana, a plant lacking homology to the RPS. Our data indicate that the palindromic element also acts as a de novo hypermethylation site in the non-repetitive genomic background of Arabidopsis, strongly suggesting a signal function of the palindromic RPS element.

Animals↗

Two classes of Tn10 transposase mutants that suppress mutations in the Tn10 terminal inverted repeat.

Tn10 transposition requires IS10 transposase and essential sequences at the two ends of the element. Mutations in terminal basepairs 6-13 confer particularly strong transposition defects. We describe here the identification of transposase mutations that suppress the transposition defects of such terminus mutations. These mutations are named "SEM" for suppression of ends mutations. All of the SEM mutations suppress more than a single terminus mutation and thus are not simple alterations of transposase/end recognition specificity. The mutations identified fall into two classes on the basis of genetic tests, location within the protein and nature of the amino acid substitution. Class I mutations, which are somewhat allele specific, appear to define a small structural and functional domain of transposase in which hydrophobic interactions are important at an intermediate stage of the transposition reaction, after an effective interaction between the ends but before transposon excision. Class II mutations, which are more general in their effects, occur at a single residue in a small noncritical amino-terminal proteolytic domain of transposase and exert their affects by altering a charge interaction; these mutations may affect act early in the reaction, before or during establishment of an effective interaction between the ends.

Codon↗

Characterization of major recognition sequences for a herpes simplex virus type 1 origin-binding protein.

To investigate early initiation events in the replication of herpes simplex virus type 1, we analyzed interactions of proteins from infected cell extracts with the small origin of herpes simplex virus type 1 (oris1). Using the mobility shift assay, we detected two origin-specific binding interactions. We characterized the more prominent interaction on both strands of the DNA duplex with DNase I protection and methylation interference assays. Protein binding protects 17 bases of DNA on each strand from DNase I. These sequences are located at the left end of the central palindrome and are shifted four bases relative to one another. On the basis of the DNase protection pattern, we believe this protein to be related to the origin-binding protein defined by Elias et al. (P. Elias, M.E. O'Donnell, E.S. Mocarski, and I.R. Lehman, Proc. Natl. Acad. Sci. 83:6322-6326, 1986). Our DNase I footprint shows both strong and weak areas of protection. The regions strongly protected from DNase I align with the essential contact residues identified by interference footprinting. Methylation interference defines a small binding domain of 8 base pairs: 5'-GTTCGCAC-3'/3'-CAAGCGTG-5'. This recognition sequence contains two inverted 5'-GT(T/G)CG-3' repeats which share a 2-base overlap; thus, the origin-binding protein probably binds to the inverted repeats as a dimer.

Base Sequence↗

The gene for the beta subunit of porcine LH: clusters of GC boxes and CACCC elements.

The gene for the beta subunit of porcine LH (LH-beta) was cloned from a genomic library constructed in EMBL3. The nucleotide sequence was determined for the entire gene transcriptional unit of porcine LH-beta in addition to 1277 and 372 bp of the 5'- and 3'-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the LH-beta gene is present as a single copy. The transcriptional unit of porcine LH-beta spanned 1107 bp and contained three exons interrupted by two introns of 326 and 289 bp. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues -16/-15 and +41/+42 were highly conserved in the rat, human and bovine LH-beta genes. In the 5'-flanking region, one TATA box and two CCAAT boxes were present. The steroid-responsive element was not found up to 1277 bases upstream of the transcription start site. The potential AP-2 factor-responsive elements appeared nine times within the sequence that was determined, and four of them were located in the 5'-flanking region. Two distal AP-2 elements were arranged in an inverted repeat forming a 16 bp palindromic sequence. This feature suggested that hypothalamic gonadotrophin-releasing hormone stimulates expression of the LH-beta gene, predominantly by a signal-transduction system with the protein kinase C cascade and a mediator, the AP-2 factor. A further characteristic feature of the porcine LH-beta gene was the presence of clusters of GC boxes and CACCC elements in the 5'-flanking region and the downstream sequence. Co-existence of these regulatory elements with other elements, such as the AP-2 element or CCAAT box, was also found. The porcine LH-beta gene shows a structure distinct from the porcine FSH-beta and common alpha genes, which are counterparts of the LH-beta gene, reflecting differential control of their synthesis during gametogenesis.

Amino Acid Sequence↗