Single Transcriptional Unit Research Articles

An unusual coding sequence from a Drosophila clock gene is conserved in vertebrates

The per locus has a fundamental involvement in the expression of biological rhythms in Drosophila. Mutations at this locus can shorten, lengthen or eliminate a variety of rhythmic activities that range from circadian behaviours, exemplified by eclosion and locomotor activities, to short-period behaviour such as the 55-s rhythm of courtship song. DNA from the per locus has been cloned, and we have used P-element-mediated DNA transformation to establish that a 7.1-kilobase(kb) HindIII fragment contains a functional copy of the gene. This transforming DNA contains a single transcription unit which gives rise to a 4.5-kb poly(A)+ RNA. Here we report the results of a search for sequences homologous to the per locus DNA in the genomic DNA of several species of vertebrates. An unusual, tandemly repeated sequence forming a portion of the 4.5-kb per transcript is homologous to DNA in chicken, mouse and man. Cloned DNAs from the mouse and Drosophila are related by long, uninterrupted tandem repetitions of the sequence ACNGGN. At the per locus, these tandem repeats are predicted to code for poly(Thr-Gly) tracts up to 48 amino acids long. These repeated sequences are also transcribed in the mouse. Several long tracts of poly(Thr-Gly) appear to be encoded by DNA cloned from the mouse.

Regulation of aspartate transcarbamoylase synthesis in Escherichia coli: analysis of deletion mutations in the promoter region of the pyrBI operon.

The catalytic and regulatory polypeptide chains of Escherichia coli aspartate transcarbamoylase are encoded by the pyrB and pyrI genes, respectively, which constitute a single transcriptional unit in the pyrBI operon. The DNA sequence immediately preceding the first structural gene, pyrB, contains a short open reading frame that could encode a 44-amino acid leader peptide and a (G+C)-rich region of dyad symmetry followed by eight thymidine residues. Synthesis of the enzyme is negatively controlled at the level of transcription depending on the cellular level of UTP, and an attenuation mechanism has been proposed to account for the 70-fold increase in pyrBI expression on pyrimidine starvation. The potential role of the dyad and eight thymidines as an attenuator was tested with a plasmid containing the promoter region of the pyrBI operon upstream of the galK coding sequence. When cells containing this plasmid, pPYRB10, were grown in a medium low in uracil, there was an 83-fold increase in galactokinase activity compared with the same cells grown at high uracil levels. This regulation is similar to that for aspartate transcarbamoylase synthesis in cells depleted of pyrimidines. Deletions constructed in the promoter region of pPYRB10 from the 3' side produced one plasmid that retained normal control of galK expression and five that exhibited greatly reduced regulation. Nucleotide sequence determination showed that the one deletion mutation that was functionally similar to the wild-type plasmid contained the entire region of dyad symmetry, including the eight thymidines. The plasmids with more extensive deletions lacked the region with dyad symmetry and the eight thymidines. One of the deletion mutants that exhibited very low levels of regulation lacks the entire sequence coding for the putative leader peptide up to the major promoter. The results demonstrating the crucial role of a 19-nucleotide sequence (from -33 to -15) support an attenuation model but indicate that other mechanisms also contribute to the regulation of the pyrBI operon.

Characterization of the Salmonella typhimurium mgl operon and its gene products.

In Salmonella typhimurium and Escherichia coli the high-affinity galactose transport system, which contains a periplasmic galactose-binding protein as an essential component, is encoded by the mgl genes. The entire mgl region of S. typhimurium is contained on a 6.3-kilobase EcoRI restriction fragment, which has been cloned into plasmid vectors. We determined the extent of the mgl region on this fragment by Tn5 mutagenesis, examination of lacZ fusions to mgl genes, and subcloning smaller restriction fragments. Polyacrylamide gel electrophoresis of protein preparations derived from strains carrying different plasmids was used to identify the mgl gene products. We conclude that the mgl operon consists of four genes that form a single transcription unit: mglB, mglA, mglE, and mglC. The mglB gene codes for galactose-binding protein (33,000 daltons), mglA codes for a membrane-bound protein of 51,000 daltons, and mglC codes for a 29,000-dalton membrane protein. The mglE product was less well characterized. Its existence was inferred from a mglE-lacZ protein fusion located between mglA and mglC. In addition, the coupled transcription-translation in vitro system indicated that mglE codes for a 21,000-dalton protein.

What the papers say: Keeping it in the family: How T cells make antigen receptors

AbstractThe last year has unveiled extensive information on the T‐cell antigen receptor genes. For both the α‐ and β‐chains of this molecule, it is clear that an expressed gene is compiled from several coding sequences dispersed along the chromosome. During T‐cell development, recombination events occur which create a single transcription unit from these dispersed elements. Such gene organization shows that the T‐cell receptor has close evolutionary links with immunoglobulins. Both types of molecule use the same genetic mechanisms to create the diversity necessary for antigen recognition.

Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region.

A genetic analysis of Agrobacterium tumefaciens chromosomal functions required for virulence was undertaken. Large Tn5-containing cosmid clones were isolated from DNA of avirulent A. tumefaciens mutants having chromosomal Tn5 insertions and exhibiting defective attachment to plant cells. The clones from several different mutants each contained overlapping segments of a 30-kilobase A. tumefaciens chromosomal region, which were physically mapped. All chromosomal Tn5 insertions leading to the avirulent, attachment-defective phenotype were localized within an 11-kilobase portion of this chromosomal virulence region. Transposon Tn3::HoHo1 (Tn3 containing lacZ) was used to simultaneously mutagenize and create lac fusions within the virulence region. This analysis demonstrated the presence of two distinct chromosomal virulence loci, which were 1.5 and 5 kilobases long; transposon insertions into these loci led to avirulence and defective attachment. The beta-galactosidase activity associated with various Tn3::HoHo1-created lac fusions indicated that the loci are transcribed in opposite directions, and complementation studies suggested that each locus consists of a single transcriptional unit. A cosmid clone of the chromosomal virulence region containing a lac fusion in the extreme 3' portion of the 5-kilobase locus was used to demonstrate that expression of this region is dependent on the presence of sequences in the 5' portion of the locus, confirming its operon-like nature.

Structure and transcription of the tRNAPro1 gene from Escherichia coli.

A 5 kbp DNA fragment containing the tRNAPro1 gene from Escherichia coli was cloned into Charon 21A phage and sequenced by the M13 DNA sequencing technique. When the cloned DNA fragment was used as a template for in vitro transcription with E. coli RNA polymerase, a tRNAPro1 precursor of 120 nucleotide residues was obtained. The tRNAPro1 gene transcribed as a single transcription unit was followed by two unusual repeating sequences, both of 108 bp. These two repeating sequences were separated by a 60 bp spacer sequence. The 5'-portion of each repeating sequence overlapped 19 bp of the 3'-terminal region of the tRNAPro1 gene just like the repeating sequences in the E. coli tRNATyr1 gene. A rho-independent termination was present in the first repeating unit.

Regulation of replication and maintenance functions of broad host-range plasmid RK2.

Replication of broad host-range plasmid RK2 depends on a cisacting vegatative replication origin oriVRK2 and the polypeptide product(s) of the trans-acting gene trfA as well as on host-specified products. The trfA gene is the second cistron in a polycistronic unit whose first cistron may be kilD, one of 4 known RK2-specified kil loci (kilA, B, C, and D) which are inhibitory for bacterial host or plasmid vector in the absence of kor functions which suppress in trans the effect of their respective kil genes. Transcription of the operon containing trfA is negatively regulated by the products of both the trfB locus (alias korD and korA) and korB. The loci, trfB and korB, are expressed from a single transcriptional unit which we propose to be negatively autoregulated by the products of both loci, although an additional, weaker and unregulated transcript may also express korB. While deletions in the oriVRK2 region have indicated the presence of copy number control elements adjacent to and possibly overlapping with the minimal oriVRK2 segment, the overriding control of copy number seems to reside in the trfB and korB loci which in conjunction appear to reduce expression of the trfA gene to levels limiting for replication. Coregulation of trfA with kil genes may indicate that kil genes play a role in plasmid maintenance other than replication.

Characterisation of a Dictyostelium discoideum DNA fragment coding for a putative tRNAValGUU gene.

A genomic DNA fragment from Dictyostelium discoideum was characterized. This DNA, although 74% d(A + T)-rich, codes for a putative tRNAValGUU. The tRNAVal gene overlaps at its 5' half with another RNA polymerase III transcription unit. This RNA polymerase III transcription unit can be folded into a tRNA-like shape and is comprised of significant amounts of invariant and semi-invariant nucleotides present in all eukaryotic tRNAs. This unit contains the two promoter blocks defined for RNA polymerase III, which are homologous to recently defined promoter elements to the extent of 76-88% (A block) and 86-93% (B block) respectively [Sharp et al. (1981) Proc. Natl Acad. Sci. USA 78, 6657-6661]. Both of the overlapping class III genes are transcribed in germinal vesicle extracts prepared from Xenopus laevis oocytes as a single transcription unit, resulting in an unusually large product compared to primary transcripts of other tRNA genes. The unit is not transcribed in HeLa extracts but it competes very strongly for transcription factor(s) under the conditions of stable transcription complex formation. Although the whole unit is transcribed, it is believed that only one functional product is formed. Therefore we define the tRNA-like structure, coded for on this class III transcription unit, as a putative tRNA 'pseudogene' meaning that, although it is transcribed by RNA polymerase III, it is not likely to mature to a functional tRNA.

Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: evidence for the existence of a single transcriptional unit

A cosmid containing the ptsH, ptsI and crr genes of Escherichia coli coding for three components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) has been isolated. The products of these genes were identified using the maxicell technique. The cloning of the PTS region as a whole allowed us to map and order ptsH,ptsI and crr genes in a 3.8-kb DNA fragment and determine thatptsI and crr are transcribed from a common promoter. They, therefore, constitute a single transcriptional unit which is likely to include also the ptsH gene. In addition, the crr gene may have a secondary promoter located inside ptsI.

Transcriptional mapping of human respiratory syncytial virus.

A transcriptional map for human respiratory syncytial virus was determined by measuring the kinetics of viral gene inactivation in response to UV irradiation. Monolayer cell cultures of respiratory syncytial virus-infected HEp-2 cells were exposed to UV light, and residual viral RNA synthesis was monitored both by gel electrophoresis and by hybridization to dot blots of cloned cDNAs of the 10 known viral genes. Target sizes for the 10 individual viral genes were calculated relative to the UV sensitivity of intracellular viral genome replication. Target size analysis indicated that the 10 viral genes were transcribed as a single transcriptional unit and that the transcription of an individual gene was dependent on the prior transcription of all the preceding genes. The order of gene transcription (with nomenclature according to encoded proteins) was determined to proceed from the promoter as follows: 14K, 11K, N, P, M, 9.5K, 36K, F, 24K, L.

Drug-induced dispersal of transcribed rRNA genes and transcriptional products: immunolocalization and silver staining of different nucleolar components in rat cells treated with 5,6-dichloro-beta-D-ribofuranosylbenzimidazole.

Upon incubation of cultured rat cells with the adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar "necklaces". We have used this experimental system, which does not inhibit transcription of the rRNA genes, to study by immunocytochemistry the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology. Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosomal protein (S1) of the small ribosomal subunit permit localization of nucleolar preribosomal particles. The results show that, under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase I and argyrophilic proteins. Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene-containing granules. We conclude that, although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits. We further propose that each RNA polymerase-positive granular unit represents a single transcription unit and that each continuous array of granules ("string of nucleolar beads") reflects the linear distribution of rRNA genes along a nucleolar organizer region. Based on the total number of polymerase I-positive granules we estimate that a minimum of 60 rRNA genes are active during interphase of DRB-treated rat cells.

Transposon Tn5 specifies streptomycin resistance in Rhizobium spp

Transposon Tn5 conferred streptomycin resistance on different strains of Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium trifolii but not on Escherichia coli. A gene (str) specifying this phenotype has been identified and localized on the physical and genetic map of Tn5. It is transcribed from the promoter of neo, the gene that encodes neomycin phosphotransferase. The str gene is downstream from neo in a single transcriptional unit, as revealed by molecular cloning of different segments of Tn5 and by cloning of the neo-str region of Tn5 downstream from a lac promoter. Fusion of the SalI-generated rightward segment of Tn5 (devoid of neo) to a part of a tetracycline resistance gene, tet, in a plasmid or downstream from a lac promoter in a plasmid resulted in significant levels of streptomycin resistance in an R. meliloti host, suggesting that the str gene product can function independent of neomycin phosphotransferase. A natural isolate of R. meliloti that does not express Tn5-associated streptomycin resistance has been identified. We have used the str of Tn5 as a genetic marker in Rhizobium spp.

Drosophila maternal and embryo mRNAs transcribed from a single transcription unit use alternate combinations of exons.

We have investigated the organization and transcription of several genes in Drosophila melanogaster which are clustered on an 18-kb cloned DNA fragment (c25) that maps at 99D on the cytogenetic map. Multiple mRNAs, transcribed from genes which lie adjacent to a ribosomal protein ( rp49 ) gene, are present during oogenesis, embryogenesis, or both. At least five mRNAs are transcribed from one of these genes ( EH8 ); three are zygotic transcripts, of which two are blastoderm stage-specific, whereas two others accumulate during oogenesis and are therefore matenal mRNAs. The complex transcription pattern of this gene indicates that alternate usage of protein-coding exons results in the production of different mRNAs with different coding capabilities during oogenesis and embryogenesis. The EH8 transcription unit is framed by genes actively expressed in the adult male fly; thus, the blastoderm stage-specific promoter may be silent although within a region of transcriptionally active chromatin.

Cloning, mapping, and expression of genes involved in the fatty acid-degradative multienzyme complex of Escherichia coli.

Two protein subunits (42,000 and 78,000 daltons) encoded by the fadAB genes form a multifunctional enzyme complex containing thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase, crotonase , epimerase, and isomerase activities (S. Pawar and H. Schulz, J. Biol. Chem. 256:3894-3899, 1981). In an attempt to characterize the structural organization and regulatory properties of these genes, a 5.2-kilobase pair fragment containing the fadAB genes has been isolated. Plasmids containing this fragment (i) complement mutations in the fadAB genes; (ii) overproduce by 10- to 50-fold thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase and crotonase ; and (iii) specify a 42,000- and a 78,000-dalton protein. The fadA gene, which encodes the 42,000-dalton protein, has been localized within the original clone to a 3.3-kilobase pair fragment. Thiolase activity, which is encoded by the 42,000-dalton protein, was not observed in the absence of the 78,000-dalton protein, suggesting that an intact complex is required for function. Transposon Tn5 insertional mutagenesis of the cloned fadAB genes has demonstrated that both fadA and fadB are transcribed as a single transcriptional unit with the direction of transcription from fadA to fadB . The molecular cloning and characterization of the fadAB region confirm the original genetic contention that the genes encoding the proteins for the multifunctional complex form an operon.

Ty1-promoted expression of aspartate transcarbamylase in the yeast Saccharomyces cerevisiae.

An entire copy of a Ty1 yeast transposon has been found inserted between two regions comprising the single transcriptional and translational URA2 units in yeast that code respectively for carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). The mutant Rev 16 was obtained from an ATCase- strain blocked by multiple nonsense mutations in the proximal CPSase region and submitted to selective pressure for recovery of the enzyme activity coded by the distal part of the gene. The inserted Ty1 has one XhoI site in both delta elements, delimiting a 5.6 kb piece of DNA that shows a classical Ty1 restriction pattern. The orientation of this sequence in URA2 is the same as in the previously described examples in which Ty1 has positive effects on the expression of adjacent genes. In this case the Ty1 is situated more than 1 kb from the URA2 region in which ATCase structural mutants have been mapped. Nevertheless, transcription of the entire sequence distal to the Ty1 is restored and has become subject to mating-type control, leading to a weak enzyme activity. Our observations are in agreement with generally accepted ideas regarding the way in which Ty1 elements affect gene expression, and additionally, represent the first example of a Ty1 -promoted reinitiation occurring in the middle of a single transcription unit.

TRNAMetf2 gene in the leader region of the nusA operon in Escherichia coli.

The promoter-proximal portion of the operon containing the Escherichia coli nusA gene has been cloned. Its nucleotide sequence shows that genes for tRNAMetf2 and a 15-kilodalton protein of unknown function precede the nusA protein gene. The sequence suggests that the three genes form a single transcription unit. Consistent with this hypothesis, purified RNA polymerase formed full-length transcripts on the cloned DNA in vitro, although transcription was frequently arrested at the intercistronic site(s) between the gene for tRNAMetf2 and the 15-kilodalton protein.

Expression of a cloned K88ac adhesion antigen determinant: identification of a new adhesion cistron and role of a vector-encoded promoter.

The determinant for the K88ac adherence antigen of porcine enterotoxigenic Escherichia coli has been cloned previously onto the vector plasmid pBR322 to form the K88ac-pBR322 hybrid plasmid pMK005 (M. Kehoe et al., Nature [London] 291:122-126). Further studies on the expression of the K88ac antigen from pMK005 are presented in this paper. Expression was found to be dependent mainly on the P1 promoter of the pBR322 vector. The natural K88ac promoter was apparently not cloned from the original parental K88ac plasmid. The P1 promoter was deleted and replaced by a DNA sequence encoding the promoter-operator region of the E. coli tryptophan (Trp) operon. Cells harboring the Trp-pMK005 hybrid plasmid expressed high levels of K88ac antigen when the Trp promoter was repressed. If the promoter was derepressed either by growing the cells in low concentrations of tryptophan or in the presence of indole acrylic acid, growth of the cells harboring the Trp-pMK005 hybrid plasmid was inhibited. A quantitative assay was used to measure the levels of K88ac antigen expressed by cells harboring different pMK005::Tn5 plasmids. All cells were found to express a reduced level of K88ac antigen, providing evidence that a single transcription unit, initiating at promoter P1 of pBR322, may be involved in the expression of the K88ac antigen. By constructing specific deletion and insertion mutants of pMK005, a fifth adhesion cistron, tentatively named adhE, was identified and mapped at the proximal end of the K88ac determinant. Although the cistron is required for high-level expression of K88ac surface-associated fimbriae, as yet no gene product has been assigned to adhE.

Visualization of a lampbrush loop-forming fertility gene in Drosophila hydei

Chromatin of D. hydei spermatocytes was spread according to Miller. One of the Y-chromosomal lampbrush loops, called the “nooses”, has been identified in the Miller spreads. It consists of a single giant transcription unit with a DNA length of more than 260 kb. The transcripts of this loop have a complex morphology. Comparison of the lampbrush loop in chromatin spreads and in ultrathin sections revealed that the transcript morphology is maintained during the spreading procedure.

Isolation of the rudimentary locus of Drosophila melanogaster

In Drosophila melanogaster the rudimentary locus encodes the first three enzymes in de novo pyrimidine biosynthesis: CPSase, ATCase and DHOase. rudimentary is located towards the proximal end of the X chromosome at the cytogenetic locus 15A1. A variety of genetic and molecular studies have suggested that all three enzymatic activities are encoded in a single transcription unit whose mRNA is translated into a multifunctional polypeptide with a molecular weight of approximately 220,000 D. In this paper we describe the isolation of a 90 kb D. melanogaster DNA segment which contains the rudimentary gene. This was achieved by screening a D. melanogaster genomic phage library with a cDNA probe homologous to the corresponding genetic unit of hamster (pyr 1–3). One of the phage recombinants homologous to this hamster probe, BS313 was found to be derived from the 15A1 region of the X chromosome. Starting from BS313 we “walked” in both directions along the X chromosome to isolate the entire rudimentary locus plus flanking DNA segments. To positively identify the rudimentary gene we in situ hybridized the various phage recombinants obtained in this walk to an inversion chromosome that has one breakpoint at 15A1 which inactivates the gene. The phage immediately adjacent to BS313 was found to span the inversion breakpoint and we were able to determine the orientation of the phage walk on the basis of this in situ hybridization result. Finally we indentify a portion of the rudimentary mRNA coding region on the basis of homology first to the hamster cDNA probe and to total Drosophila RNA and second, to a 7.3 kb long poly A+ transcript which is found both in adult flies and in embryos.

Nucleotide sequence for the catalytic domain of colicin E3 and its immunity protein. Evidence for a third gene overlapping colicin.

We have determined the nucleotide sequence of a segment of Co1E3 DNA coding for the carboxyl-terminal, catalytic peptide of colicin E3 and for the immunity protein. The end of the colicin E3 gene is separated from the beginning of the immunity gene by a nine-basepair intercistronic region, suggesting the two genes are expressed as a single transcriptional unit. The immunity gene is expressed, however, in E. coli strains containing Co1E3-pBR322 hybrid plasmids deleted for the 5'-end of the colicin gene. The DNA sequence also contains an unexpected open reading frame (ORF). This ORF is contained within the colicin gene and is in the +1 reading frame with respect to that gene. Plasmids containing the ORF, directed the synthesis of an 11 kilodalton protein in a cell-free, transcription-translation system.