Transcriptional Region Research Articles

The highly edited orf206 in Oenothera mitochondria may encode a component of a heme transporter involved in cytochrome c biogenesis.

A highly transcribed region in Oenothera mitochondria codes for a reading frame (orf206) which shows high homology to the Marchantia encoded mitochondrial open reading frame orf277 and is also conserved in the mitochondrial genomes of Arabidopsis thaliana and Daucus carota. Transcripts of orf206 are modified by cytidine to uridine changes in 46 positions by RNA editing, affecting 30% of all cytidines and 15% of the total encoded amino acids. This ORF is cotranscribed with an upstream reading frame and with the downstream rps 14 gene. The orf206 deduced protein shows high similarity to polypeptides which are proposed to be part of an ABC-type heme transporter involved in cytochrome c biogenesis in Bradyrhizobium and Rhodobacter.

Pet, a small sequence distal to the pregenome cap site, is required for expression of the duck hepatitis B virus pregenome

We have found that transcription of the pregenome of an avian hepadnavirus, duck hepatitis B virus (DHBV), is dependent on the presence of a small element in the 5' transcribed region of the pregenome-encoding sequence. This element, which we have named pet (positive effector of transcription), exerts its effect in cis in a position and orientation-dependent manner, suggesting that it may function as part of the nascent pregenome transcript. The requirement for pet depends on the presence in the transcription unit of a region of the DHBV genome located upstream of the envelope promoters, which specifically suppresses transcription of templates lacking pet. In the presence of this region, deletion of pet activates transcription from downstream promoters, suggesting that pregenome transcription complexes fail to reach the downstream promoters. In vitro transcription experiments support the model that pet is required for transcription elongation on the DHBV template. We speculate that pet is required to suppress transcription termination during the first passage of pregenome transcription complexes through a viral termination region on the circular viral DNA.

Effects of promoter, intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts

Various chimaeric promoter regions coupled to the uidA beta-glucuronidase gene were evaluated for transient expression strength following electroporation into sugar-cane (monocot) and carrot (dicot) protoplasts. Multiple enhancer elements increased expression in sugar-cane, by up to 400-fold for the artificial Emu promoter relative to the CaMV 35S promoter. The relative expression strengths of promoters varied substantially between the species. Sugar-cane also differed in some respects from previously tested species in the family Poaceae. For example, in sugar-cane the nopaline synthase and CaMV 35S promoters were of equivalent strength, and insertion of Adh1 intron 1 into the 5' transcribed region decreased expression strength.

In Drosophila Kc cells 20-OHE induction of the 60C beta3 tubulin gene expression is a primary transcriptional event.

In Drosophila Kc cells, the 60C beta3 tubulin transcription unit, whose expression is induced by 20-hydroxyecdysone (20-OHE), has the same structure as in Drosophila. This gene is characterized by an unusual 5' intron of regulating importance, by an alternatively spliced second intron and by a long 3' transcribed but untranslated region. This gene codes for two beta3 tubulin isoforms with one amino acid difference. We have established that beta3 tubulin gene expression is transcriptionally regulated by the steroid hormone in a time and hormonal concentration-dependent fashion, without requirement of protein synthesis. This implies that this transcriptional induction is a primary event and that this gene is probably a direct target for the 20-OHE receptor.

Cloning and characterization of a gene encoding the endopolygalacturonase of Fusarium moniliforme

The N-terminus and several internal tryptic peptides of endo polygalacturonase purified from Fusarium moniliforme were sequenced. The amino acid sequences of the N-terminus and of one of the tryptic peptides were used to design redundant oligonucleotides. A DNA fragment of 288 bp, encoding part of the polygalacturonase gene, was amplified by the polymerase chain reaction (PCR) using the oligonucleotides as primers and, as a template, cDNA synthesized from poly(A) + RNA extracted from mycelium grown on pectin as a sole carbon source. The PCR-generated fragment was utilized as a probe to screen a genomic and a cDNA library of F. moniliforme . Genomic and cDNA copies of the endo polygalacturonase gene were isolated and sequenced. A cloned 1272 bp cDNA consisted of a single open reading frame encoding 359 amino acids (including the entire 349 aa mature protein and part of the N-terminal signal peptide), the 3' transcribed untranslated region (173 nt), and the poly(A) tail. The nucleotide sequence of the genomic endo polygalacturonase gene was interrupted by four short introns (50–54 bp) and revealed the presence of a 24 amino-acid signal peptide. By RNAse protection experiments, multiple sites of initiation for the transcription of the endo polygalacturonase gene were demonstrated, from 33 to 45 nt downstream from the putative TATA box.

A human DNA replication origin: localization and transcriptional characterization.

A single-copy 13.7 kb human DNA region (L30E) located on Ch. 19 p13.3 contains an origin of DNA replication in myeloid HL-60 cells. The origin was localized, by means of quantitative PCR within approximately 3000 bp, in a highly transcribed region containing at least two closely spaced genes with the same polarity of transcription, one encoding lamin B2 and the other an unidentified protein. The origin region overlaps an undermethylated "CpG island" at the 5'-end of the second transcription unit. A binding site (CACGTG) for basic helix-loop-helix (bHLH) DNA binding proteins such as USF/MLTF or MYC-MAX was located by DNase I footprinting analysis in the promoter of the second gene. DMSO differentiation of HL-60 cells, that completely shuts off replication, also drastically reduces the transcription of L30E region. On the other hand such treatment does not modify the methylation pattern of the CpG island and does not abolish the DNase I protection of the bHLH binding site.

The suppressor of Hairy-wing binding region is required for gypsy mutagenesis.

We undertook a deletional analysis of the gypsy retrotransposon in order to determine which sequences of the element are required for its mutagenic effect. We show that a phenotype indistinguishable from that of y2 flies can be generated by transforming y- flies with a construct containing the yellow gene and a gypsy element located at the same insertion site in yellow as found in y2 flies. When flies are transformed with similar constructs in which increasing amounts of the 5' transcribed untranslated region of gypsy have been removed, either a partial y2 revertant or a completely revertant phenotype is obtained. These results yield direct proof that the region of gypsy to which the su(Hw) protein binds is required for the generation of mutant phenotypes by this retrotransposon.

Alternative chromatin structure at CpG islands

Actively transcribed chromatin is structurally different from bulk inactive chromatin. It has been difficult to define the molecular basis of the difference, however, because purified fractions of active chromatin were not available. We have overcome this problem by releasing oligonucleosomes from the nonmethylated CpG-rich islands (CpG islands) of HeLa cell nuclei using restriction endonucleases. Since CpG islands very often include the promoters and 5′ transcribed regions of genes, they represent a model for the “active” chromatin structure. CpG island chromatin differs in three respects from bulk chromatin prepared in the same way: histone H1 is present in very low amounts; histones H3 and H4 are highly acetylated; and nucleosome-free regions are present. Except for the latter regions, the average nucleosomal spacing is similar to that of bulk chromatin.

Characterization of a highly transcribed DNA region of herpesvirus of turkeys

A highly transcribed DNA region of turkey herpesvirus (HVT) codes for multiple species of RNA. Although the RNA is transcribed from both strands of DNA, no overlapping complementary transcripts have been detected. The complete nucleotide sequence corresponding to the cDNA of one of the HVT transcripts was determined. This sequence is highly homologous to the nucleotide sequence of Marek's disease virus antigen gp57–65-coding gene and appears to represent the corresponding HVT-coding gene.

Association of adenovirus DNA transcribed activity with nuclear matrix of host cells.

With gentle cell extraction techniques, various DNA components in the HeLa cells after 6 h of adenovirus infection have been obtained. Adenovirus, early transcribed regions (E2a, E1b) and a late transcribed region (L2) were used as probes in Southern hybridization, respectively. The experiment showed that only actively transcribed adenovirus DNA fragments would tightly bind to the nuclear matrix of host cells. We inferred that the nuclear matrix of host cells plays an important role in viral DNA transcription.

A functional mitochondrial ATP synthase proteolipid gene produced by recombination of parental genes in a petunia somatic hybrid.

A novel ATP synthase subunit 9 gene (atp9) was identified in the mitochondrial genome of a Petunia somatic hybrid line (13-133) which was produced from a fusion between Petunia lines 3688 and 3704. The novel gene was generated by intergenomic recombination between atp9 genes from the two parental plant lines. The entire atp9 coding region is represented on the recombinant gene. Comparison of gene sequences indicate that the 5' transcribed region is contributed by an atp9 gene from 3704 and the 3' transcribed region is contributed by an atp9 gene from 3688. The recombinant atp9 gene is transcriptionally active. The location of the 5' and 3' transcript termini are conserved with respect to the parental genes, resulting in the production of hybrid transcripts.

On the specificity and effects on transcription of P-element insertions at theyellowlocus ofDrosophila melanogaster

Fourteen P-element insertion mutants at the yellow (y) gene of D. melanogaster have been analysed by restriction mapping and DNA sequencing. The distribution of the insertion sites is nonrandom, with a preferred target site in the 5'-transcribed but non-translated leader of the gene. Transcription analysis indicates that P-elements inserted into the transcribed region of y can terminate y transcription. While the great majority of the P-element insertions into the preferred target site abolish y activity, this loss of activity is not due to disruption of the insertion site since imprecise excision events which do not restore the wild type sequence at the insertion site can restore y function. We further demonstrate that a mutant caused by a 221bp P-element insertion into the 5' transcribed but non-translated region, which show partial y activity, produces a larger mutant transcript consistent with it being a product of transcriptional readthrough. We present results which suggest that imprecise excision events which restore (or partially restore) y activity can occur by deleting P-element internal sequences which normally act to terminate y transcription.

Molecular evolution and nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH).

The nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH) are presented. The evolution of these genes among higher plants is characterized by a transition mutation bias of about 2:1 and by rates of synonymous and nonsynonymous substitution which are much lower than similar rates for genes from other sources. This is consistent with the notion that the plastid genome is evolving conservatively in primary sequence. Yet, the mode and tempo of sequence evolution of these and other plastid-encoded coupling factor genes are not the same. In particular, higher rates of nonsynonymous substitution in atpE (the gene for the epsilon subunit of CF1) and higher rates of synonymous substitution in atpH in the dicot vs. monocot lineages of higher plants indicate that these sequences are likely subject to different evolutionary constraints in these two lineages. The 5'- and 3'-transcribed flanking regions of atpA and atpH from maize, wheat and tobacco are conserved in size, but contain few putative regulatory elements which are conserved either in their spatial arrangement or sequence complexity. However, these regions likely contain variable numbers of "species-specific" regulatory elements. The present studies thus suggest that the plastid genome is not a passive participant in an evolutionary process governed by a more rapidly changing, readily adaptive, nuclear compartment, but that novel strategies for the coordinate expression of genes in the plastid genome may arise through rapid evolution of the flanking sequences of these genes.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region.

To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized. Starting with the previously cloned ADE1 gene (J. C. Crowley and D. B. Kaback, J. Bacteriol. 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization. The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1). The CDC15 gene was located by isolating plasmids from a YCp50 S. cerevisiae genomic library that complemented the cdc15-1 mutation. S. cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization. The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene. This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1. CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S. cerevisiae plasmid vectors. Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15. In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element. Notably, a higher than average proportion of the DNA examined was transcribed into RNA.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region.

To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized. Starting with the previously cloned ADE1 gene (J. C. Crowley and D. B. Kaback, J. Bacteriol. 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization. The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1). The CDC15 gene was located by isolating plasmids from a YCp50 S. cerevisiae genomic library that complemented the cdc15-1 mutation. S. cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization. The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene. This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1. CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S. cerevisiae plasmid vectors. Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15. In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element. Notably, a higher than average proportion of the DNA examined was transcribed into RNA.

Differential expression of mouse beta/goat beta c, mouse beta/goat beta F, and mouse beta/goat epsilon II hybrid globin genes in murine erythroleukemia cells.

We assembled three hybrid beta-globin genes by fusing the mouse beta-major promoter and initial transcribed region to one of three goat beta-like globin gene bodies: beta c (preadult), beta F (fetal), or epsilon II (embryonic). Thymidine kinase (tk)-deficient murine erythroleukemia (MEL) cells were cotransformed with one of these constructs and a separate plasmid bearing the tk gene. Half of the 24 cell lines containing either the mouse beta/goat beta c or mouse beta/goat beta F genes expressed the transferred genes at significant levels; in many cases the hybrid genes were, like the endogenous beta-globin genes, inducible with dimethyl sulfoxide. We obtained 13 cell lines containing the mouse beta/goat epsilon II hybrid gene, 6 of which were cotransfected with a mouse beta/human beta fusion gene known to function in MEL cells. In contrast to the results with the other fusion genes, the mouse beta/goat epsilon II hybrid was very poorly expressed: in two separate experiments, 0 of 13 and 2 of 13 lines showed significant mouse beta/goat epsilon II RNA levels after induction. In all these lines the endogenous mouse beta and cotransfected mouse beta/human beta genes were expressed. As an initial test of possible reasons for the inactivity of the mouse beta/goat epsilon II hybrid, we recloned this fusion gene into a tk-bearing plasmid, adjacent to the tk gene. Of 12 cell lines transformed with this plasmid, 11 produced mouse beta/goat epsilon II RNA; in 6 cases the expression was both strong and dimethyl sulfoxide inducible.

Differential expression of mouse beta/goat beta c, mouse beta/goat beta F, and mouse beta/goat epsilon II hybrid globin genes in murine erythroleukemia cells.

We assembled three hybrid beta-globin genes by fusing the mouse beta-major promoter and initial transcribed region to one of three goat beta-like globin gene bodies: beta c (preadult), beta F (fetal), or epsilon II (embryonic). Thymidine kinase (tk)-deficient murine erythroleukemia (MEL) cells were cotransformed with one of these constructs and a separate plasmid bearing the tk gene. Half of the 24 cell lines containing either the mouse beta/goat beta c or mouse beta/goat beta F genes expressed the transferred genes at significant levels; in many cases the hybrid genes were, like the endogenous beta-globin genes, inducible with dimethyl sulfoxide. We obtained 13 cell lines containing the mouse beta/goat epsilon II hybrid gene, 6 of which were cotransfected with a mouse beta/human beta fusion gene known to function in MEL cells. In contrast to the results with the other fusion genes, the mouse beta/goat epsilon II hybrid was very poorly expressed: in two separate experiments, 0 of 13 and 2 of 13 lines showed significant mouse beta/goat epsilon II RNA levels after induction. In all these lines the endogenous mouse beta and cotransfected mouse beta/human beta genes were expressed. As an initial test of possible reasons for the inactivity of the mouse beta/goat epsilon II hybrid, we recloned this fusion gene into a tk-bearing plasmid, adjacent to the tk gene. Of 12 cell lines transformed with this plasmid, 11 produced mouse beta/goat epsilon II RNA; in 6 cases the expression was both strong and dimethyl sulfoxide inducible.

DNA sequence of an immediate-early gene (IEmRNA-5) of herpes simplex virus type I.

We describe a 2560 base pair herpes simplex virus type 1 (HSV-1) DNA sequence containing the entire immediate-early mRNA-5 (IEmRNA-5) gene. The 3' and 5' termini of IEmRNA-5 were mapped within this DNA sequence by single-strand specific endonuclease protection experiments. The IEmRNA-5 gene contains DNA sequences from both the unique (Us) and reiterated (TRs/IRs) regions of the HSV-1 DNA short component and is interrupted by a single intron mapping in TRs/IRs. A search of the transcribed DNA sequence revealed no initiator codon within TRs/IRs. The first ATG was located 6 bases into Us sequences and this reading frame (316 codons) was also observed in the 3' transcribed region. The oligonucleotide sequences adjacent to the IEmRNA-5 termini are discussed in relation to those of the HSV-1 thymidine kinase gene and other genes transcribed by RNA polymerase II.

The organization and transcription of the galactose gene cluster of Saccharomyces

The transcriptional activity of a kb † † Abbreviations used: kb, base × 10 3. region of Saccharomyces DNA centered on three galactose-inducible sequences has been examined. An in vivo-generated gal7 −, gal10 −, gal1 − mutation, gal▽ (isolated by D. C. Hawthorne), is a 7 kb deletion that overlaps the map positions of all three inducible RNAs. These three RNAs correspond to the transcripts of the GAL7-GAL10-GAL1 gene cluster located on chromosome II. Gene assignments have been inferred by comparison of the published protein sizes and the experimentally determined messenger RNA lengths. The physically assigned gene order, GAL7-GAL10-GAL1, agrees with the genetically established order. The GAL7 and GAL10 genes (which encode galactose-1-phosphate uridylyl transferase and uridine diphosphogalactose-4-epimerase, respectively) are transcribed from the same DNA strand. The GAL1 gene (which encodes galactokinase) is transcribed from the complementary DNA strand such that its 5′ end is 0.65 kb from the 5′ end of the GAL10 gene. These three genes are normally transcribed into at least six discrete RNA species in the strains examined. Two additional GAL1 transcripts have been detected at the non-permissive temperature in the yeast rna1-1 “nuclear transport” mutant. None of these eight transcripts contains detectable intervening sequences. Transcription of the GAL1 gene results in the presence of five detectable RNA molecules (1.65, 1.75, 4.5, 5.5 and 6.5 to 7 kb), the two longest having only been detected in the rna1-1 mutant at the restrictive temperature. These five transcripts differ only at their 3′ ends. The 5′ ends of these RNAs are indistinguishable by S 1 nuclease mapping procedures. The two smallest RNAs are only slightly larger than required to encode galactokinase. GAL7 and GAL10 gene sequences are found in 1.25 and 2.25 kb RNA molecules. respectively, and are separated by about 0.65 kb. These separate GAL7 and GAL10 RNAs arc only approximately 200 bases longer than required to encode their respective proteins. These two genes are also transcribed together into a large RNA molecule (4.15 kb), 0.65 kb longer than the sum of the lengths of the separate GAL7 and GAL10 messenger RNAs. The 5′ end of this molecule cannot be distinguished from the 5′ end of the GAL10 mRNA. Its 3′ end is very near or identical to the 3′ end of the GAL7 mRNA. The yeast rna1-1 mutant, deficient in nuclear transport (and/or RNA processing), accumulates the three largest GAL1 transcripts at the non-permissive temperature. Under these conditions, the rna1-1 mutant also accumulates the large RNA containing both GAL7 and GAL10 gene sequences as well as the 1.25 kb GAL7 RNA. These data suggest the presence of a promoter for the transcription of the GAL1 gene, an adjacent promoter for the transcription of GAL10 and/or both GAL10 and GAL7, and a third promoter for the GAL7 gene. The 28 kb of cloned DNA sequences extend beyond the gene cluster and contain at least four transcribed regions that are not likely to be related to the GAL gene cluster. All of the DNA in the cluster region itself is unique, although repeated DNA sequences are located at the extreme left edge of the cloned region.