Single Transcriptional Unit Research Articles

Regulation of transcription and promoter mapping of the structural genes for nitrogenase (nifHDK) of Azospirillum brasilense Sp7.

Transcription of the structural genes for nitrogenase (nifHDK) in Azospirillum brasilense Sp7 was analysed using Northern blots of total RNA extracted from cultures grown under nitrogen-fixing conditions. Hybridization with an internal nifH probe revealed two transcripts, a major one (by concentration) of 1.1 kb corresponding to nifH and a minor one of 5.6 kb corresponding to nifHDK. Hybridization with nifD or nifK probes revealed the minor transcript of 5.6 kb. This confirms that the nifHDK genes are organized as a single transcription unit and suggests regulation at the level of termination of transcription. The complete nucleotide sequence of nifH was established and the DNA region upstream of the initiation codon was analysed for transcription and translation signals. The nifH open reading frame (ORF) is preceded by an NtrA-dependent promoter and two elements homologous to upstream activator sequences (UAS) required for NifA-mediated activation in other diazotrophs. Promoter mapping with S1 nuclease revealed two start sites located 10 bp and 40 bp downstream of the NtrA-dependent promoter.

Transcription map of the B genome component of tomato golden mosaic virus and comparison with A component transcripts

In a previous study, the bipartite genome of tomato golden mosaic virus (TGMV) was shown to be transcribed into at least six polyadenylated RNAs ( G. Sunter, W. E. Gardiner, and D. M. Bisaro, 1989, Virology 170, 243–250). Two of these, a 1.3-kb complementary sense and a 0.9-kb viral sense transcript, were mapped to the B genome component of this geminivirus. The results of more detailed primer extension and S, nuclease protection experiments presented here define the limits of the single transcription unit corresponding to the 0.9-kb RNA which spans the BR1 open reading frame (ORF). The data also demonstrate that complementary sense TGMV RNAs are more complex than indicated by our earlier studies. Analysis of the 1.3-kb BL1-specific RNA indicates that it is actually a family of distinct transcripts with different start sites. Three transcripts have 5′ ends that map near the common region of DNA B and all of these start sites lie upstream of the BL1 ORE Similar analysis of the 1.6-kb complementary sense AL1 RNA indicates that a complex set of transcripts also map to the analogous region of genome component A. Four transcripts have 5′ ends that map near the common region but only one of these start sites is upstream of the initiation codon for the AL1 open reading frame (ORF). None of the transcripts appear to be processed. The possible significance of multiple transcripts in these regions of the TGMV genome is discussed, and the common region-proximal transcription units of the A and B genome components are compared.

Cloning and Genetic Analysis of Six Pyrroloquinoline Quinone Biosynthesis Genes in Methylobacterium organophilum DSM 760

After EMS mutagenesis, mutants of Methylobacterium organophilum DSM 760 unable to synthesize pyrroloquinoline quinone (PQQ) were selected among mutants which did not utilize methanol but were still able to use methylamine as growth substrate. Six different pqq genes (pqqA to pqqF) were identified by complementation analysis. The genes pqqA to pqqD, cloned in a single R′ plasmid, were grouped in a 3·9 kb DNA fragment. The genes pqqA and pqqB belonged to a single transcription unit independent from the adjacent gene pqqC. The gene pqqD was contained in a short DNA segment of approximately 0·1 kb, separated from pqqC by a region with no apparent role in PQQ biosynthesis. Two other genes were identified:pqqE, which was closely linked to pqqD; and pqqF, located approximately 19 kb from the other genes. Directed mutagenesis by marker exchange provided chromosomal insertion mutations of these genes in M. organophilum. Attempts to express the pqq genes in two heterologous hosts, Escherichia coli and Pseudomonas testosteroni, were unsuccessful, and no plasmid containing all of the pqq genes was isolated.

Fibrillar center distribution in nucleoli of PHA-stimulated human lymphocytes

We have utilized acidic toluidine blue staining for RNA and immunofluorescence staining for RNA polymerase 1 to visualize the distribution of fibrillar centers (FCs) in nucleoli of PHA-stimulated human lymphocytes. At 0 h, there is a single large fibrillar center in each nucleolus which splits into smaller and more numerous FCs until the number of FCs reaches five, the number of nucleolus organizers in normal haploid human cells. With time, each FC then "unwinds" to form linear arrays of smaller FCs until the maximum number of FCs approaches the ribosomal gene copy number of 200 at 48 h in culture. It is hypothesized that in the most active state, each nucleolar FC visualized by RNA polymerase 1 staining actually represents a single transcription unit and the distance between adjacent FCs is occupied by the nontranscribed spacer region. We conclude that the number of fibrillar centers per nucleolus can be used as a direct quantitative measure of nucleolar transcriptional activity.

Isolation and molecular analysis of the maize P locus

The maize P locus is involved in the synthesis of a red flavonoid pigment in the pericarp, cob and other floral tissues. The tissue-specific pattern of expression of certain P alleles suggests that P may be a complex locus, with more than one functional unit. The P-VV allele, which specifies variegated pericarp and variegated cob, however, shows that insertion and excision of the transposable element Ac affects both pericarp and cob expression as though cob and pericarp pigmentation are controlled by a single gene. Using Ac as a transposon tag, we have isolated 34 kb of genomic DNA from the P-VV and P-RR allele. The cloned DNA contains two 5.8 kb cross-hybridizing regions, in direct orientation relative to each other, separated by 6.6 kb of intervening DNA. A sequence motif of 250 bp is repeated at three locations within the cloned region: once within each of the 5.8 kb repeats, and once outside the 5.8 kb repeats. DNA fragments flanking the Ac element detect five transcripts in RNA from wild type (P-RR) that are absent from mutant (P-VV) tissues. To localize the transcribed sequences, DNA probes spanning the 34 kb of cloned DNA were used in Northern analysis of RNA from mutant and wild-type kernels. The results suggest the presence of a single transcriptional unit located primarily within the DNA between the 5.8 kb repeats. The five RNAs transcribed from this region may be formed by alternative splicing. The size of the P gene derived from the length of the transcribed region seems much smaller than the gene size estimated from Ac-induced P-VV mutations.

Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis

Cytochrome aa3 is one of two terminal oxidase complexes in the Bacillus subtilis electron transport chain. A novel genetic strategy was devised which permitted the isolation of B. subtilis mutants lacking cytochrome aa3 by selection for streptomycin-resistant clones which failed to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Two mutations were studied intensively. Spectroscopic examination showed that each mutant lacked cytochrome aa3; they were also asporogenous and unable to grow on lactate as the sole carbon and energy source. These mutations were mapped to a locus designated ctaA, located at 127 degrees between pyrD and metC on the B. subtilis chromosome. Both ctaA mutations were closely linked by transformation to the pycA locus. The ctaA locus and a portion of the pycA locus were cloned from a B. subtilis integration library constructed in Escherichia coli. A recombinant plasmid containing a 4.0-kilobase insert of B. subtilis DNA could transform both ctaA mutants to CtaA+. Gene disruption and complementation experiments with subcloned fragments revealed that the ctaA locus consisted of a single transcriptional unit about 1.35 kilobase pairs in size. The nucleotide sequence of the ctaA transcriptional unit contains a single open reading frame capable of coding for a protein with a predicted molecular weight of 34,065. The predicted protein is extremely hydrophobic, with several probable membrane-spanning domains. No sequence similiarity was found between ctaA and the highly conserved procaryotic and mitochondrial oxidase polypeptides. Cloning and sequence analysis of two ctaA mutations revealed that one allele is a nonsense mutation in the carboxy terminus and the other is a missense mutation in the amino terminus; this indicates that the pleiotropic phenotype conferred by each mutation was caused by loss of CtaA or of its activity. Genetic evidence suggests that the ctaA gene product is required as an accessory protein in the genetic expression, posttranslational biogenesis, or both, of the cytochrome aa3 complex and during an early stage of sporogenesis.

Organization of the flaFG gene cluster and identification of two additional genes involved in flagellum biogenesis in Caulobacter crescentus.

In Caulobacter crescentus, mutations have been isolated in more than 30 flagellar genes (fla, flb, and flg) which are required in the cell cycle event of flagellum biogenesis. The flaF and flaG mutations and two newly identified mutations, flbT and flbA (P.V. Schoenlein and B. Ely, J. Bacteriol. 171:000-000, 1989), have been localized to the flaFG region. In this study, the genetic and physical organization of this region was analyzed, using the cloned 4.0-kilobase flaFG region in the recombinant plasmid pPLG727. Plasmid pPLG727 complemented flaF, flaG, flbA, and flbT mutations. Further complementation studies with pPLG727 derivatives indicated that flaF and flbT are unique but overlapping transcription units, whereas flbA and flaG constitute a single transcription unit. To determine the direction of transcription of the putative flbA-flaG operon, the promoterless chloramphenicol transacetylase gene was inserted into various positions in the flbA-flaG region, and merodiploid strains containing these transcriptional fusions were assayed for gene function and expression of chloramphenicol resistance. These studies showed that transcription proceeds from flbA to flaG. To confirm the complementation analysis, Southern analyses were performed on chromosomal DNAs isolated from strains containing insertion and deletion mutations. Taken together, these studies defined the relative gene order at one end of the flaYG flagellar gene cluser as flgL-flaF-flbT-flbA-flaG.

Mechanisms of genetic variability in Halobacterium halobium: the purple membrane and gas vesicle mutations

Several phenotypic variants of Halobacterium halobium arise spontaneously at extremely high frequencies (up to 1%) and are readily identified by inspection of bacterial colonies. Two mutant types, those lacking the buoyant gas vesicles or the photosynthetic purple membrane, have been studied in detail by phenotypic and molecular genetic analysis. In the wild-type NRC-1 strain, the bop gene, encoding the purple membrane protein bacterio-opsin, is found on the bacterial chromosome, while the gas vesicle protein genes, gvpA and gvpC, are present on pNRC100, a multicopy plasmid of approximately 150 kilobase pairs. The gvpA and gvpC genes are on a single transcription unit, while the major bop mRNA is monocistronic. Essentially all of the purple membrane deficient mutants contain insertion sequence (IS) elements into or upstream of the bop gene. Two elements, ISH 1 and ISH 2, account for most (80-90%) of the purple membrane mutations, but at least three other elements, ISH 23, ISH 26, and ISH S1, have also been implicated. The gas vesicle mutants are more heterogeneous, with many displaying partial phenotypes. Three major classes of gas vesicle mutants are distinguishable: class I and class III mutants are the result of large deletions in pNCR100; however, while class I mutants are partially gas vesicle deficient and contain a correspondingly reduced number of gvpAC operon copies, class III mutants contain no detectable copies of the gas vesicle genes and are essentially completely gas vesicle deficient. Class II mutants, like the purple membrane mutants, contain IS elements into or upstream of the gas vesicle genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure differences between proteins C1 and C2 of HeLa 40S nuclear ribonucleoprotein particles.

Partial acid cleavage, comparative HPLC tryptic peptide mapping and amino acid sequencing of the C1 and C2 proteins of HeLa heterogeneous nuclear ribonucleoprotein (hnRNP) particles demonstrate that proteins C1 and C2 differ in primary structure by the presence of a 13 amino acid insert sequence in C2. This C2 insert sequence occurs after either glycine 106 or serine 107 in C1. The additional 13 amino acids that are present in C2 account for the observed molecular weight difference between the C1 and C2 hnRNP proteins on SDS polyacrylamide gel electrophoresis. Because C1 and C2 appear identical except for the 13 residue insert and because the 3' and 5' untranslated regions of the corresponding mRNAs also appear to be the same (Swanson et al., Mol. Cell. Biol. 7: 1731-1739), it is possible that both polypeptides are produced from a single transcription unit through an alternative splicing mechanism.

Trans splicing in trypanosomes — archaism or adaptation?

In trypanosomes, a single transcription unit usually covers several protein-coding genes. The primary transcript is cut up by trans-splicing and polyadenylation machineries to generate individual mature mRNAs. All nuclear mRNAs acquire the same capped 39 nucleotide sequence at their 5′ end as a consequence of the trans-splicing event. Trans splicing is used in the synthesis of some mRNAs in nematodes and chloroplats. These unusual systems are clearly related to cis-splicing systems, but it remains an intriguing question whether they are merely exotic offshoots of cis splicing or archaic remnants of cis-splicing progenitors.

Identification of different β amyloid cDNAs cloned from the brain of a patient with sporadic alzheimer's disease

Neurofibrillary tangles and senile plaques, two neuropathological markers of Alzheimer's disease, may both contain peptide fragments derived from the β amyloid protein. Human β amyloid peptide precursor cDNAs have been isolated from normal foetal and adult brain libraries. In peripheral tissue and cultured cells, a novel precursor containing a protease inhibitor domain has been cloned. A cDNA library from the cerebral cortex of a patient with sporadic Alzheimer's disease was constructed and several clones coding for the β amyloid peptide precursor were isolated cDNAs containing two types of insertion coding for a serine protease inhibitor domain were identified. The use of another polyadenylation site available in the 3′-untranslated region of the mRNA was observed. These results indicate that, in one patient with Alzheimer's disease, different RNA species coding for the β amyloid peptide precursor arise by alternative splicing of a single transcriptional unit, and use different polyadenylation sites.

Nucleotide sequence of the rodA gene, responsible for the rod shape of Escherichia coli: rodA and the pbpA gene, encoding penicillin-binding protein 2, constitute the rodA operon.

The rodA gene, which is responsible for the rod shape of Escherichia coli, was located 5 nucleotides downstream of another rod-shape-determining gene, pbpA, encoding penicillin-binding protein 2. The coding region for the RodA protein was 1,110 base pairs in length. Two plasmids, carrying a rodA-lacZ gene fusion with and without the pbpA promoter upstream of the gene fusion, were constructed. On the basis of the difference between the expression levels of the beta-galactosidase activity dependent on and independent of the pbpA promoter, we concluded that the pbpA and rodA genes constitute a single transcriptional unit called the rodA operon.

DNA recognition element required for PUF-I mediated cell-type-specific transcription of the rat prolactin gene

The cell-type-specific transcription of the prolactin gene in vitro is mediated through the interaction of prolactin upstream factor I (PUF-I) with a 28 basepair region of the gene promoter (-63 to -36) which contains an 18 bp A+T-rich imperfect palindrome (-63 to -46). Base substitutions were introduced into 16 of the 18 palindromic residues by targeted saturation mutagenesis. The GH3 binding and in vitro transcription assays of the mutated promoters showed that base substitutions within the 5'-ATATTCA-3' sequence located at -52 to -46 were detrimental to PUF-I binding and its cell-type-specific transcriptional enhancement activity. Transcription assays of the mutated promoters performed with several nonpituitary-derived extracts demonstrated that a distal TATA box located from -59 to -53 promotes initiation at -27. Thus, the cell-type-specific cis-acting element required by PUF-I for DNA recognition is immediately adjacent to a general TATA sequence. Base substitutions that decreased +1 transcription and PUF-I binding concomitantly increased -27 initiation of RNA in vitro. We suggest that PUF-I binding in pituitary cells potentiates +1 transcription and represses alternative TATA box activity for initiation events occurring at -27. This is the first known report of a eukaryotic DNA binding protein that has both an activator and repressor activity for a single transcription unit.

The molecular genetics of Enhancer of split, a gene required for embryonic neural development in Drosophila.

In Drosophila, the very first steps in neurogenesis appear to be controlled by a small group of zygotically acting genes termed the neurogenic loci. Mutations in any of these genes result in a misrouting of epidermal lineages into the neural pathway. Morphological and molecular studies suggest that the correct ectodermal differentiation is mediated by a cell-cell interaction mechanism and that at least some of the neurogenic loci are involved in this mechanism. The molecular analyses of the neurogenic loci Notch and Delta revealed that the putative gene products are large transmembrane proteins with homology to mammalian epidermal growth factor. We describe here a molecular analysis of Enhancer of split [E(spl)], a third neurogenic locus, which displays striking genetic interactions with both Notch and Delta, suggesting a close functional relationship of the respective gene products. We provide evidence for a single genetic complementation group corresponding to a single transcription unit which is necessary for wild-type E(spl) function. P-element-mediated transformation indicates that this transcription unit includes functions associated with both the dominant E(spl)D mutation and the recessive visible allele groucho, and is necessary for the correct differentiation of the embryonic nervous system.

Daughterless, a Drosophila gene essential for both neurogenesis and sex determination, has sequence similarities to myc and the achaete-scute complex

daughterless (da) has multiple functions in Drosophila embryonic development: maternal da activity is necessary for proper sex determination, and zygotic da activity is necessary for formation of the peripheral nervous system. We have cloned the region containing da and have found that five recessive lethal da mutations map to a single transcription unit. A predicted protein product of this transcription unit has sequence similarities with the oncogene myc, with the gene MyoD1, which is involved in myoblast determination, and with the Drosophila achaete-scute complex, which is involved in neuronal precursor determination. The role of da as a gene controlling cell determination in multiple developmental pathways is discussed.

Rhodobacter capsulatus puf operon encodes a regulatory protein (PufQ) for bacteriochlorophyll biosynthesis.

Biosynthesis of the photochemical apparatus by purple nonsulfur photosynthetic bacteria is known to be inhibited by molecular oxygen and high light intensity. Polypeptides that bind bacteriochlorophyll (BChl) to form the light-harvesting I (LH-I) and reaction-center (RC) complexes are encoded by a single transcriptional unit termed the puf operon. In this investigation we demonstrate that the first structural gene in the puf operon (pufQ) of Rhodobacter capsulatus encodes a protein that is required for BChl biosynthesis and that there exists a linear relationship between the amount of pufQ expression and the level of BChl synthesis. Protein sequence similarity exists between PufQ and the region of RC polypeptides that are known to bind BChl and quinone. These observations suggest that pufQ may regulate BChl biosynthesis by a "carrier polypeptide" mechanism as originally proposed by Lascelles.

A genetic-physical map of the Rhodobacter capsulatus carotenoid biosynthesis gene cluster

We used the interposon mutagenesis technique to map a cluster of Rhodobacter capsulatus genes that are responsible for the biosynthesis of carotenoids, membrane pigments that protect living organisms from photooxidations. Fifteen interposons define 7 genes (crtA, crtI, crtB, crtC, crtD, crtE and crtF) in an approximately 8 kb of the chromosome. This cluster is flanked by genes affecting bacteriochlorophyll biosynthesis. Based on the analysis of the phenotypes of the insertional mutants and the results of complementation analyses, we present a genetic and physical map of this region. Our data indicate that, with the possible exception of crtI and crtB, all genes analyzed are organized as single transcriptional units. We used nuclease S1 protection analysis to study the influence of oxygen on the regulation of expression of 4 genes (crtA, crtI, crtC and crtE). Under anaerobic conditions, mRNA levels for crtA, crtC and crtE are elevated and a new crtE transcript is detected.

Characterization of the promoter region of the Bacillus subtilis spoIIE operon.

Mutations that define the spoIIE locus of Bacillus subtilis block sporulation at an early stage and recently were shown to prevent the proteolytic processing of sigma E (sigma 29) into its active form, an event that is believed to control critical changes in gene expression during the second hour of development. By taking advantage of two Tn917-mediated insertional mutations in spoIIE, we have cloned DNA spanning the locus. Gene disruption experiments with subcloned fragments transferred to integrational vectors revealed that the locus consisted of a single transcription unit about 2.5 kilobase pairs in size. Transcriptional lacZ fusions were used to show that expression of this transcription unit initiated at 1.5 h after the end of log-phase growth and depended upon the products of all spo0 loci. Expression was directed by a single promoter whose position was determined by high-resolution S1 protection mapping. A deletion analysis of the promoter region was also carried out, with novel integrational vectors based on derivatives of coliphage M13. The results indicated that a region of DNA extending from 183 to 118 base pairs upstream from the start point of transcription was required for full activity of the spoIIE promoter. The presumptive RNA polymerase-binding region of the promoter exhibited striking similarity to the spoIIG promoter and featured perfect but unusually spaced -10 and -35 consensus sequences for sigma A (sigma 43)-associated RNA polymerase.

Characterization and mapping of the agrocinopine-agrocin 84 locus on the nopaline Ti plasmid pTiC58

Overlapping segments of pTiC58 inserted into cosmid vectors were used to characterize the agrocinopine-agrocin 84 locus from the nopaline/agrocinopine A and B Agrobacterium tumefaciens strain C58. All of the clones conferring agrocin 84 sensitivity on agrobacteria also conferred uptake of agrocin 84 and agrocinopines A and B. Transposon Tn3-HoHo1 insertion mutations of one such clone were generated that simultaneously abolished agrocin 84 sensitivity and transport of agrocinopines A and B and agrocin 84. Such insertions were found to cluster within a 4.4-kilobase region. Analysis of beta-galactosidase activity in these insertion mutants suggested a single transcriptional unit regulated at the transcriptional level by agrocinopines A and B. The smallest DNA fragment subcloned from the region to confer all three activities was 8.5 kilobases long. This subclone was still properly regulated, indicating that the regulatory gene is closely linked to the locus. The data are consistent with a single operon encoding catabolism of agrocinopines A and B and conferring sensitivity to agrocin 84. Based on these results, we support the locus name acc, for agrocinopine catabolism.

Cloning and sequencing of the genes for Shiga toxin from Shigella dysenteriae type 1.

The structural genes for Shiga toxin, designated stx A and stx B, were cloned from Shigella dysenteriae type 1 3818T, and a nucleotide sequence analysis was performed. Both stx A and stx B were present on a single transcriptional unit, with stx A preceding stx B. The molecular weight calculated for the processed A subunit was 32,225, while the molecular weight of the processed B subunit was 7,691. Comparison of the nucleotide sequences for Shiga toxin and Shiga-like toxin I (SLT-I) from Escherichia coli revealed that the genes for Shiga toxin and SLT-I were greater than 99% homologous; three nucleotide changes were detected in three separate codons of the A subunits. Only one of these codon differences resulted in a change in the amino acid sequence: a threonine in Shiga toxin at position 45 of the A subunit compared with a serine in the corresponding position in SLT-I. Furthermore, Shiga toxin and SLT-I had identical signal peptides for the A and B subunits, as well as identical ribosome-binding sites, a putative promoter, and iron-regulated operator sequences. These findings indicate that Shiga and SLT-I are essentially the same toxin. Southern hybridization studies with total cellular DNA from several Shigella strains and internal toxin probes for SLT-I and its antigenic variant SLT-II showed that a single fragment in S. dysenteriae type 1 hybridized strongly with the internal SLT-I probe. Fragments with weaker homology to the SLT-I probe were detected in S. flexneri type 2a but no other shigellae. No homology between the Shiga-like toxin II (SLT-II) probe and any of the Shigella DNAs was detected. Whereas SLT-I and SLT-II are phage encoded, no phage could be induced from S. dysenteriae type 1 or other Shigella spp. tested. These results suggest that the Shiga (SLT-I) toxin genes responsible for high toxin production are present in a single copy in S. dysenteriae type 1 but not in other shigellae. The findings further suggest that SLT-II genes are absent in shigellae, as are toxin-converting phages.