Single Transcriptional Unit Research Articles

Six nodulation genes of nod box locus 4 in Rhizobium meliloti are involved in nodulation signal production: nodM codes for d-glucosamine synthetase

The nucleotide sequence of the nod box locus n4 in Rhizobium meliloti was determined and revealed six genes organized in a single transcriptional unit, which are induced in response to a plant signal such as luteolin. Mutations in these genes influence the early steps of nodule development on Medicago, but have no detectable effect on Melilotus, another host for R. meliloti. Based on sequence homology, the first open reading frame (ORF) corresponds to the nodM gene and the last to the nodN gene of Rhizobium leguminosarum. The others do not exhibit similarity to any genes sequenced so far, so we designated them as nolF, nolG, nolH and nolI, respectively. We found that the n4 locus, and especially the nodM and nodN genes, are involved in the production of the root hair deformation (Had) factor. NodM exhibits homology to amidotransferases, primarily to the D-glucosamine synthetase encoded by the glmS gene of Escherichia coli. We demonstrated that in E. coli the regulatory gene nodD together with luteolin can activate nod genes. On this basis we showed that nodM complemented an E. coli glmS- mutation, indicating that nodM can be considered as a glmS gene under plant signal control. Moreover, exogenously supplied D-glucosamine restored nodulation of Medicago by nodM mutants. Our data suggest that in addition to the housekeeping glmS gene of R. melioti, nodM as a second glmS copy provides glucosamine in sufficient amounts for the synthesis of the Had factor.

Differential accumulation of plastid transcripts encoding photosystem II components in the mesophyll and bundle-sheath cells of monocotyledonous NADP-malic enzyme-type C4 plants

As a first step in understanding the differential expression of the plastid-encoded photosystem II (PSII) genes in mesophyll and bundle-sheath cells, we have used RNA blotting techniques to investigate the transcript patterns of these genes in three NADP-malic enzymetype C4 species: Zea mays L., Sorghum bicolor (L.) Moench and Pennisetum americanum (L.) Leeke. Our comparison showed that in all three species the relative levels of transcripts encoding PSII proteins were diminished in bundle-sheath cells. No major differences, either in abundance or in the processing pathways, could be detected for transcripts encoding subunits of the PSI and ATP-synthase complexes. The transcript profiles of the psbB and psbD/C transcription units were particularly striking. These operons were of heterogeneous composition, i.e. they encode PSII subunits as well as proteins or RNAs which are involved in different functional entities. The transcript patterns of the psbB and psbD/C transcription units were complex and characterized by multiple, partially overlapping RNAs. Our analysis showed that the relative levels of the oligocistronic PSII transcripts derived from these transcription units with the exception of psbH were selectively reduced in bundlesheath cells. In contrast, RNAs carrying the non-PSII components were present in similar quantities in the two cell types. The data demonstrate that segmental RNAs within one single transcription unit can accumulate to different degrees. Regulatory mechanisms which may explain this expression behaviour are discussed.

In vivo transcriptional pattern in theinfC operon ofBacillus stearotbermophilus

By Northern blot and primer extension analyses it was shown that in Bacillus stearothermophilus the genes infC, rpmI and rplT constitute a single transcriptional unit; the promoter and the transcriptional start-point used in vivo were identified and the half-life of the transcript (1.2 min) was determined. No indication of multiple initiation sites nor of differential stability of different regions of the transcript was found. The results suggest that Escherichia coli and B. stearothermophilus have a different pattern of transcription around the infC gene cluster and that differential translational efficiency within the infC-rpmI-rplT transcriptional unit accounts for the different levels of IF3, L35 and L20 expression. The rare AUU initiation codon is the only strictly conserved element of the several peculiar transcriptional and translational features found in E. coli infC. Upon changing this codon to AUG, we found a ca. 30-fold increased expression of B. stearothermophilus infC, which is similar to that previously found with E. coli infC (i.e. 40-fold), and provided evidence that regulation of infC expression through its rare AUU initiation codon might be a general phenomenon in bacteria.

Isolation and characterization of the Drosophila retinal degeneration B (rdgB) gene.

Retinal degeneration-B (rdgB) mutants of Drosophila melanogaster undergo rapid light-induced retinal degeneration. We conducted a molecular characterization of the rdgB gene to examine the nature of the gene product. Through the isolation and analysis of X-ray-induced rdgB alleles, the cytogenetic position of the gene was determined to be the 12C1 salivary region. Genomic DNA corresponding to this region was isolated by a chromosomal walk. The chromosomal aberrations associated with the three X-ray-induced rdgB alleles were shown to be within a 5-kb genomic region. A single transcription unit was affected by the alleles, identifying it as the rdgB gene. RNA-RNA Northern hybridization indicated the rdgB gene transcribed five mRNAs ranging in size from 3.9 to 9.5 kb. These mRNAs were expressed in adult heads, but not detected in bodies. Analysis of RNA isolated from wild-type and eyes absent heads indicated that rdgB mRNA expression was not restricted to the retina. DNA sequence analysis of the transcription unit revealed an open reading frame capable of encoding a 116-kD transmembrane protein. The deduced protein shows no overall homology to previously described proteins, but has sequences in common with proposed functional domains of Ca(2+)-ATPase.

Trypanosoma equiperdum minicircles encode three distinct primary transcripts which exhibit guide RNA characteristics.

The mitochondrial DNA of trypanosomes is composed of maxicircle and minicircle DNAs catenated into a network, called the kinetoplast. Maxicircles encode proteins and RNAs necessary for mitochondrial assembly. Minicircles encode small transcripts which are believed to serve as guide RNAs in the process of RNA editing of maxicircle transcripts. Trypanosoma equiperdum minicircles contain three transcription units which produce three distinct transcripts. The genes for these transcripts are flanked by imperfect 18-bp repeats separated by approximately 110 bp. The transcripts have a 5' triphosphate, indicating that they are primary transcripts. Minicircle transcription initiates at a purine within a conserved sequence, 5'-AYAYA-3', where Y is a pyrimidine, 32 bp from the upstream inverted repeat, suggesting that the repeats may function in transcript initiation. Transcripts from a single minicircle transcription unit range in size from 55 to 70 nucleotides. This size heterogeneity within a single sequence class is due to the variable length of nontemplated uridine residues composing a 3' tail. The size range and heterogeneous polyuridylate 3' end of the minicircle transcripts appear to be conserved features and may be related to transcript function.

Trypanosoma equiperdum minicircles encode three distinct primary transcripts which exhibit guide RNA characteristics.

The mitochondrial DNA of trypanosomes is composed of maxicircle and minicircle DNAs catenated into a network, called the kinetoplast. Maxicircles encode proteins and RNAs necessary for mitochondrial assembly. Minicircles encode small transcripts which are believed to serve as guide RNAs in the process of RNA editing of maxicircle transcripts. Trypanosoma equiperdum minicircles contain three transcription units which produce three distinct transcripts. The genes for these transcripts are flanked by imperfect 18-bp repeats separated by approximately 110 bp. The transcripts have a 5' triphosphate, indicating that they are primary transcripts. Minicircle transcription initiates at a purine within a conserved sequence, 5'-AYAYA-3', where Y is a pyrimidine, 32 bp from the upstream inverted repeat, suggesting that the repeats may function in transcript initiation. Transcripts from a single minicircle transcription unit range in size from 55 to 70 nucleotides. This size heterogeneity within a single sequence class is due to the variable length of nontemplated uridine residues composing a 3' tail. The size range and heterogeneous polyuridylate 3' end of the minicircle transcripts appear to be conserved features and may be related to transcript function.

Quantitative determination of rDNA transcription units in vertebrate cells

The adenosine analogue 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) unravels the compact nucleoli to necklace-like structures when applied to living cells. The nucleolar beads contain RNA polymerase I (RPI) and argyrophilic proteins, both properties considered to be characteristic of ribosomal gene activity. Each granule is supposed to represent a single transcription unit consisting of an actively transcribing gene and its RPI complex. Indirect immunofluorescence with anti-RPI antibodies was used to determine the number of transcription units in DRB-treated cells of some representative mammals, marsupials, birds, and amphibians. We estimate that 45 to 145 rRNA genes are transcriptionally active in vertebrate fibroblasts, depending on the species. Nucleolar transcriptional activity does not correlate with the total number of rRNA genes. During in vitro aging of fibroblasts, the number of transcription units appears to remain unchanged. Different cell types of one same organism show varying numbers of transcription units, reflecting their differential metabolic activity. A particular situation exists in phytohemagglutinin-stimulated lymphocytes. In the course of nucleolar activation, the number of transcription units is increased considerably, implying that formerly inactive rRNA genes are recruited for transcription. The opposite phenomenon is observed during spermatogenesis. With the diploid spermatocytes developing into haploid spermatids, the transcriptionally active rRNA genes decrease in number until rRNA synthesis is completely blocked.

Overlapping mRNA transcripts of photosynthesis gene operons in Rhodobacter capsulatus

The crtEF, bchCA, and puf operons of the facultative phototrophic bacterium Rhodobacter capsulatus encode gene products that are necessary for the formation of various components of the photosynthetic apparatus. The crtEF operon encodes two enzymes involved in the biosynthesis of carotenoids, the bchCA operon codes for two enzymes of the bacteriochlorophyll biosynthetic pathway, and the puf operon encodes four pigment-binding polypeptides as well as two polypeptides with less well understood functions. These three operons are adjacent to one another on the chromosome and are transcribed in the same direction. We present the results of RNA blotting and S1 nuclease protection end-mapping experiments which provide direct evidence that the mRNA transcripts of these three operons overlap. Therefore, it is likely that the crtEF, bchCA, and puf operons can be expressed as a single transcriptional unit, although RNA polymerase may initiate transcription at any of several promoters.

Alternative splicing is an efficient mechanism for the generation of protein diversity: Contractile protein genes as a model system

Alternative splicing has emerged in recent years as a widespread device for regulating gene expression and generating protein diversity. Its analysis has provided some mechanistic understanding of this form of gene regulation and, in addition, has provided new insights into some fundamental aspects of splicing. This mode of regulation is particularly prevalent in muscle cells, where genes such as troponin T are able to generate up to 64 different isoforms from a single transcriptional unit. Alternative splicing has the potential to raise the coding capacity of the small multigene families that code for the contractile proteins so that several million structurally different sarcomeres can be generated. The mammalian α-tropomyosin gene has proved particularly useful for the analysis of the mechanisms involved in this type of regulation. In particular, the mutually exclusive splicing of exons 2 and 3 has provided answers about the processes involved in the three main regulatory steps: (a) establishment of mutually exclusive behavior; (b) the elements involved in setting up the default pattern of splicing, and (c) the switch from the default to the regulated splicing pattern in some cell types.

Larvicidal properties of the 42 and 51 kilodaltonBacillus sphaericus proteins expressed in different bacterial hosts: Evidence for a binary toxin

The 51 kilodalton (kDa) and the 42 kDa larvicidal proteins ofBacillus sphaericus 2362 appear to reside on a single transcriptional unit. Constructs containing the genes for both of these proteins or for only the 51 kDa or the 42 kDa protein were inserted into the appropriate expression vectors and the proteins over-produced inEscherichia coli JM105, andBacillus sphaericus strain 718 and SSII-1. In all of these organisms the 51 kDa and 42 kDa proteins were not toxic alone but were toxic in combination. These results are interpreted to indicate that theBacillus sphaericus 2362 larvicide is a binary toxin in that both the 51 and the 42 kDa proteins are required for toxicity.

Thyrotropin activates both the cyclic AMP and the PIP 2casades in CHO cells expressing the human cDNA of TSH receptor

The effect of thyrotropin (TSH) on cyclic AMP accumulation, phosphatidylinositol bisphosphate (PIP 2) hydrolysis and [Ca 2+] i rise has been studied in CHO cells stably transfected with human TSH receptor (hTSHR) cDNA. In human thyroid slices, TSH activates these two intracellular cascades with a higher affinity for the adenylate cyclase activation (from 0.1 to 1 mU/ml TSH) than for phospholipase C activation (from 1 to 10 mU/ml TSH). The CHO cells transfected with the recently cloned cDNA of human TSH receptor respond in the same way to TSH. They respond between 0.1 and 1 mU/ml TSH for cyclic AMP accumulation and between 1 and 10 mU/ml TSH for inositol monophosphate (IP 1) increase. In these same cells, TSH 10 mU/ml, but not forskolin (10 μM), or dibutyryl cyclic AMP (100 μM), clearly enhances intracellular calcium concentration ([Ca 2+] i). Our results demonstrate unequivocally that a single transcription unit has the potential to encode receptor molecules coupled to both cascades.

Molecular analysis of the Escherichia coli K5 kps locus: identification and characterization of an inner‐membrane capsular polysaccharide transport system

The complete nucleotide sequence has been determined of a region of the Escherichia coli K5 antigen gene cluster postulated to encode functions for the translocation of capsular polysaccharide across the inner membrane. This revealed two genes, designated kpsM and kpsT, organized in a single transcriptional unit. Analysis of the predicted amino acid sequence of the KpsM and KpsT proteins indicates that they may function as dual components in a polysaccharide export system analogous to the periplasmic binding protein-dependent transport systems of Gram-negative bacteria. We propose that the KpsT protein acts as an energizer, coupling ATP hydrolysis to the transport process mediated by the KpsM protein. Extensive sequence homology between the KpsM and KpsT proteins and the products of the bexB and bexA genes present in the capsulation (cap) locus of Haemophilus influenzae, indicates that a common mechanism for the export of polysaccharide across the inner membrane may exist in these two micro-organisms.

The molecular structure of the Corynebacterium glutamicum threonine synthase gene

The minimal region encoding the Corynebacterium glutamicum threonine synthase structural gene and its promoter was mapped by deletion analysis and complementation of the C. glutamicum thrC allele to a 1.6 kb region of the recombinant plasmid pFS80. The nucleotide sequence of this and flanking DNA was determined. The transcription and translation start points were identified by S1 mapping analysis and amino-terminal protein sequencing, respectively. The thrC gene encodes a 54481-Dalton polypeptide product. Translation of the thrC mRNA initiates only six nucleotides downstream from transcription. The length of the mRNA transcript is consistent with a single gene transcription unit. The C. glutamicum thrC gene is expressed independently of the other threonine-specific genes hom and thrB.

Nitrous oxide reductase from denitrifying Pseudomonas stutzeri

Nitrous oxide (N2O) respiration by the denitrifying bacterium Pseudomonas stutzeri requires the synthesis of the multi-copper enzyme N2O reductase, nosZ, the structural gene for this enzyme, is part of a DNA region of approximately 8 kbp that carries several essential genes. Insertional mutagenesis of the region downstream of nosZ generates apoenzyme-synthesizing strains, which argues for the existence of functions for copper acquisition or copper processing, or both, for N2O reductase. The relevant DNA region of approximately 3.2 kbp was sequenced and found to consist of three genes, nosDFY, presumably within a single transcriptional unit. Cellular concentration, copper content, and location of the N2O reductase protein were studied with mutants which were affected in the three genes. Interactions of the deduced gene products among each other and with the cytoplasmic membrane appear to be analogous to those of the components of osmotic-shock-sensitive bacterial transport systems. The 33.8-kDa product of the nosF gene belongs to the family of nucleotide-binding proteins [C. Higgins et al. (1986) Nature 323, 448-450]. Its amino acid sequence shows two canonical nucleotide-binding motifs, and the positional identity of amino acids to members of this family is around 30%. The 29.4-kDa product of the nosY gene is a hydrophobic protein with six predicted transmembrane helices and an export signal. The 48.2-kDa product of the nosD gene is a periplasmic component; it carries an export signal and is a hydrophilic protein. N2O reductase itself is a periplasmic enzyme. Our results provide evidence for an auxiliary system of Cu processing and suggest its involvement in the periplasmic biosynthesis of the Cu centers of N2O reductase.

Identification of a locus that regulates multiple functions in Pseudomonas solanacearum

When Pseudomonas solanacearum K60 carries a multicopy plasmid containing cosmid clone pE6C (from the wild-type strain K60) or pBE6 (from the nonpathogenic strain B1), several phenotypic changes are observed, including the following: loss of virulence, reduced extracellular polysaccharide production, and increased polygalacturonase activity. Both cosmids contain a common 8-kilobase DNA region that is required for the phenotype shift. Saturation mutagenesis of pBE6 with Tn3-gus suggested that a single transcriptional unit of at least 1 kilobase is responsible for the phenotype shift. In maxicell assays, subclones containing this transcriptional unit expressed a single protein of about 25 kilodaltons.

Biosynthesis of peptide precursors and protease inhibitors using new constitutive and inducible eukaryotic expression vectors

A series of expression vectors has been constructed as based on the pML derivative of pBR322. The eukaryotic transcription units employ various promoters followed by polycloning sites for 3–9 commonly used restriction enzymes and are completed by the SV40 polyadenylation sequence. In 4 of the vectors, designed for co-transfection or transient expression studies, only a single transcription unit containing either a constitutive or an inducible promoter was incorporated. The human ubiquitin (UbC) promoter was used as a strong constitutive promoter, while the mouse metallothionein promoter and the promoter of the long terminal repeats of the mouse mammary tumor virus were used as inducible promoters. Another vector contained an additional transcription unit encoding a eukaryotic selection marker, the neomycin resistance encoding gene. The vectors were used in CHO cells and in neuroendocrine CA77 cells to synthesize peptide precursors, protease inhibitors and a protease. It is shown that these vectors are very efficient for the constitutive and inducible expression of nucleotide sequences in both transient and stable transfections of eukaryotic cells.

Complex RNA maturation pathway for a chloroplast ribosomal protein operon with an internal tRNA cistron.

We have studied the expression of a large chloroplast ribosomal protein operon from Euglena gracilis that resembles the Escherichia coli S10 and spc ribosomal protein operons. We present evidence that 11 ribosomal protein genes, a tRNA gene, and a new locus, orf214/orf302, are expressed as a single transcription unit. The primary transcript also contains at least 15 group II and group III introns. Gene-specific probes for each ribosomal protein gene, orf214/orf302, and for trnl hybridized to a common pre-mRNA of an estimated size of 8.3 kilobases. This is the RNA size predicted for a full-length transcript of the entire operon after splicing of all 15 introns. Polycistronic ribosomal protein mRNAs accumulated primarily as spliced hepta-, hexa-, penta-, tetra-, tri-, and dicistronic mRNAs, which were presumed to arise by stepwise processing of the 8.3-kilobase pre-mRNA. A novel finding was the cotranscription of the trnl gene as an internal cistron within the ribosomal protein operon. Several combined mRNA/tRNA molecules, such as the pentacistronic rpl5-rps8-rpl36-trnl-rps14, were characterized. The occurrence of the orf214/orf302 is a unique feature of the Euglena operon, distinguishing it from all chloroplast and prokaryotic ribosomal protein operons characterized to date. The orf214/orf302 are not similar to any known genes but are cotranscribed with the ribosomal protein loci and encode stable RNA species of 2.4, 1.8, and 1.4 kilobases.

DNA sequences of genes encoding Acinetobacter calcoaceticus protocatechuate 3,4-dioxygenase: evidence indicating shuffling of genes and of DNA sequences within genes during their evolutionary divergence

The DNA sequence of a 2,391-base-pair HindIII restriction fragment of Acinetobacter calcoaceticus DNA containing the pcaCHG genes is reported. The DNA sequence reveals that A. calcoaceticus pca genes, encoding enzymes required for protocatechuate metabolism, are arranged in a single transcriptional unit, pcaEFDBCHG, whereas homologous genes are arranged differently in Pseudomonas putida. The pcaG and pcaH genes represent separate reading frames respectively encoding the alpha and beta subunits of protocatechuate 3,4-dioxygenase (EC 1.13.1.3); previously a single designation, pcaA, had been used to represent DNA encoding this enzyme. The alpha and beta protein subunits appear to share common ancestry with each other and with catechol 1,2-dioxygenases from A. calcoaceticus and P. putida. Marked conservation of amino acid sequence is observed in a region containing two histidyl residues and two tyrosyl residues that appear to ligate iron within each oxygenase. In some regions within the aligned oxygenase sequences, DNA sequences appear to be conserved at a level beyond the extent that might have been demanded by selection at the level of protein. In other regions, divergence of DNA sequences appears to have been achieved by substitution of DNA sequence from one genetic segment into another. The results are interpreted to be the consequence of sequence exchange by gene conversion between slipped strands of DNA during evolutionary divergence; mismatch repair between slipped strands may contribute to the maintenance of DNA sequence in divergent genes.

The organization and evolution of transfer RNA genes inMycoplasma capricolum

The genes for presumably all the tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been cloned and sequenced. There are 30 genes encoding 29 tRNA species. This number is the smallest in all the known genetic systems except for mitochondria. The sequences of 9 tRNA genes of them have been previously reported (1-3). Twenty-two genes are organized in 5 clusters consisting of nine, five, four and two genes (2 sets), respectively. The other eight genes exist as a single transcription unit. All the tRNAs are encoded each by a single gene, except for the occurrence of two tRNA(Lys)(TTT) genes. The arrangement of tRNA genes in the 9-gene cluster, the 5-gene cluster, the 4-gene cluster and one of the 2-gene clusters reveals extensive similarity with a part of the 21-tRNA gene cluster and/or the 16-tRNA gene cluster in Bacillus subtilis, respectively. The results suggest that the present M. capricolum tRNA genes have evolved from large tRNA gene clusters in the ancestral Gram-positive bacterial genome common to M. capricolum and B. subtilis, by discarding genes for redundant as well as non-obligate tRNAs, so that all the codons may be translated by as small a number of tRNAs as possible.

TheTrypanosoma bruceiprotein phosphatase gene: polycistronic transcription with the RNA polymerase II largest subunit gene

We have previously described the trypanosomal gene encoding the largest subunit of RNA polymerase II (RNAP II) and found that two almost identical genes are encoded within the Trypanosoma brucei genome. Here we show by Southern analyses that the 5' breakpoint between both loci is located approximately 7.5 kb upstream of the RNAP II genes. Northern analyses revealed that the 5' duplicated segment contains at least four other genes, which are transcribed in both bloodstream and procyclic trypanosomes. The gene located immediately upstream of the RNAP II gene in both loci was characterized by sequence analyses. The deduced amino acid sequences show a high degree of similarity to the catalytic subunit of protein phosphatase class 1 (PP1) genes. S1 mapping provided strong evidence in support of the fact that the PP1 and RNAP II genes belong to a single transcription unit.